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Association of human breast cancer CD44-/CD24- cells with delayed distant metastasis

  1. Xinbo Qiao
  2. Yixiao Zhang
  3. Lisha Sun
  4. Qingtian Ma
  5. Jie Yang
  6. Liping Ai
  7. Jinqi Xue
  8. Guanglei Chen
  9. Hao Zhang
  10. Ce Ji
  11. Xi Gu
  12. Haixin Lei
  13. Yongliang Yang
  14. Caigang Liu  Is a corresponding author
  1. Department of Oncology, Shengjing Hospital, China Medical University, China
  2. Dapartment of Urology, Shengjing Hospital, China Medical University, China
  3. Department of Breast Surgery, Liaoning Cancer Hospital and Institute, Cancer Hospital of China Medical University, China
  4. Department of General Surgery, Shengjing Hospital, China Medical University, China
  5. Institute of Cancer Stem Cell, Cancer Center, Dalian Medical University, China
  6. Center for Molecular Medicine, School of Life Science and Biotechnology, Dalian University of Technology, China
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Cite this article as: eLife 2021;10:e65418 doi: 10.7554/eLife.65418

Abstract

Tumor metastasis remains the main cause of breast cancer-related deaths, especially delayed breast cancer distant metastasis. The current study assessed the frequency of CD44-/CD24- breast cancer cells in 576 tissue specimens for associations with clinicopathological features and metastasis and investigated the underlying molecular mechanisms. The results indicated that higher frequency (≥19.5%) of CD44-/CD24- cells was associated with delayed postoperative breast cancer metastasis. Furthermore, CD44-/CD24-triple negative breast cancer (TNBC) cells spontaneously converted into CD44+/CD24-cancer stem cells (CSCs) with properties similar to CD44+/CD24-CSCs from primary human breast cancer cells and parental TNBC cells in terms of stemness marker expression, self-renewal, differentiation, tumorigenicity, and lung metastasis in vitro and in NOD/SCID mice. RNA sequencing identified several differentially expressed genes (DEGs) in newly converted CSCs and RHBDL2, one of the DEGs, expression was upregulated. More importantly, RHBDL2 silencing inhibited the YAP1/USP31/NF-κB signaling and attenuated spontaneous CD44-/CD24- cell conversion into CSCs and their mammosphere formation. These findings suggest that the frequency of CD44-/CD24- tumor cells and RHBDL2 may be valuable for prognosis of delayed breast cancer metastasis, particularly for TNBC.

Introduction

Breast cancer is the most prevalent malignancy in women and its incidence is increasing globally, especially in developed countries (Bianchini et al., 2016; Siegel et al., 2020). Different treatment strategies, such as surgical resection, hormone therapy, targeted therapy, radiation therapy, and chemotherapy, have greatly improved survival of breast cancer patients (Bray et al., 2018; Burstein et al., 2014; Early Breast Cancer Trialists' Collaborative G, 2015; Khan et al., 2012; Saini et al., 2012). To date, clinical strategies for breast cancer treatment remain suboptimal. Although continuous treatment with tamoxifen for 10 years has reduced cancer recurrence and mortality of patients with luminal A breast cancer, a significant proportion of patients may be over-treated (Bianchini et al., 2016; O'Conor et al., 2018). Hence, it is urgently needed to discover novel biomarkers to predict treatment effectiveness and to improve treatment success and prognosis of breast cancer patients. Furthermore, the available therapies can also lead to a considerable number of patients at a risk to develop a delayed breast cancer metastasis, which occurs even 20–40 years after breast cancer diagnosis (Sharma, 2018). Notably, breast cancer metastasis occurring 5–8 years after initial surgical resection has become a significant cause of cancer relapse, progression, and poor survival in patients (Nishimura et al., 2013); thus, further researches on its molecular mechanisms and gene alterations may help identify novel biomarkers and targets for development of therapeutic strategies to effectively control breast cancer metastasis and progression.

Indeed, tumor metastasis is a multistep process, during which, cancer cells escape from the primary site, migration into a neighboring or distant tissues, extravasation, survival, and colonization, leading to the formation of new tumor nodules at a secondary site (Drabsch and ten Dijke, 2011; Klein, 2008; Scott et al., 2012; Syn et al., 2016). The rate-limiting step of cancer metastasis is cancer cell colonization and proliferation at the secondary site, because the initial metastatic cancer nodule usually lacks an efficient vasculature to provide sufficient nutrients to support cancer cell growth. Thus, the newly arrived tumor cells may grow to a certain size in the new and harsh microenvironment and undergo growth arrest in that organ. However, once they regain their proliferative ability, delayed metastasis will occur (Langley and Fidler, 2007). Molecularly, CD44+/CD24- breast cancer cells from primary breast tumors are associated with distant metastasis (Abraham et al., 2005), and these cells display potent motility and invasiveness (Liu et al., 2010), similar to chemoresistance cancer stem cells (CSCs) (Velasco-Velázquez et al., 2011). Previous studies have shown that CD44+/CD24- breast CSCs may be a dominant factor for the relapse of triple negative breast cancer (TNBC), due to their potent self-renewal and differentiation capacities (Geng et al., 2014; Wang et al., 2014). Indeed, injection with about 50 breast CSCs can induce a solid tumor mass in immunocompromised mice (Chaffer et al., 2011; Iliopoulos et al., 2011). Thus, the number of breast CSCs in the secondary site may affect the efficient formation of early metastatic nodules, and breast CSCs are commonly prone to be resistant to chemotherapy (De Angelis et al., 2019). Moreover, previous studies have reported that the differentiated cancer cells can spontaneously convert into CSCs to renew the CSC pool in breast cancer, pancreatic cancer and sarcomas, resulting in chemoresistance (Gruber et al., 2016; Kim et al., 2015; Ye et al., 2018). Thus, the dormant CD44-/CD24- breast cancer cells that have previously been colonized in the metastatic site may be able to spontaneously convert into CD44+/CD24- breast CSCs to regain their potent proliferative ability and drug resistance, resulting in delayed breast cancer metastasis. Accordingly, it is reasonable to hypothesize that the frequency of CD44-/CD24- cells in human tumor specimens may be useful in the prediction of delayed breast cancer metastasis.

The current study aimed to explore the molecular mechanisms by which CD44-/CD24- cell conversion into CSC promotes delayed breast cancer metastasis. First, a retrospective analysis of CD44-/CD24- breast cancer cells in tissue specimens from patients enrolled from three academic medical centers was performed to investigate the potential associations between the frequency of CD44-/CD24- cells and postoperative tumor metastasis. Next, the spontaneous CD44-/CD24- cell conversion into CD44+/CD24- CSCs was tested and the biological functions of the converted CSCs were analyzed in vitro and in vivo. The results may provide novel insights into the role of CD44-/CD24- tumor cells in delayed breast cancer metastasis and into the potential use of CD44-/CD24- cells as a biomarker to predict survival and metastasis in breast cancer patients. These findings also suggest that RHBDL2 may be a novel therapeutic target for the future studies.

Materials and methods

Patients and tissue specimens

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Paraffin-embedded surgical tissue samples were collected from 576 breast cancer patients, who underwent breast cancer surgery between June 2005 and April 2013 in China Medical University Affiliated Hospital (Shenyang, Liaoning, China), Liaoning Cancer Hospital (Shenyang, Liaoning, China), and Dalian Municipal Central Hospital Affiliated to Dalian Medical University (Dalian, Liaoning, China). The patients were diagnosed with invasive breast cancer histologically, according to the World Health Organization (WHO) breast cancer classifications, 4th edition (Tan et al., 2015) and classified, according to breast cancer TNM staging (Li et al., 2012). The demographic, clinicopathological and follow-up data of all patients were collected from their medical records or via telephone interview. The inclusion criteria were: surgical treatment for breast cancer; complete information regarding clinicopathological characteristics; and complete follow-up data. Disease-free survival (DFS) was defined as the time from the date of surgery to the date of distant metastasis, while overall survival (OS) was defined as the time from the date of surgery to the date of death. The current study was approved by the Ethics Committee of all three hospital review boards (Project identification code: 2018PS304K, date on 03/05/2018), and each participant signed an informed consent form before being included in the study.

Immunofluorescence staining

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Paraffin-embedded tissue blocks of 576 breast cancer patients were prepared to construct a tissue microarray (Edge and Compton, 2010). The levels of CD44 and CD24 expression in the tissue microarray sections were assayed using a double immunofluorescence staining. Briefly, individual sections (4 µm) on the tissue microarray glass slides that had been pre-coated with (3-aminopropyl) triethoxysilane solution were deparaffinized, rehydrated, and subjected to regular antigen retrieval. The sections were blocked with 10% fetal bovine serum (FBS, Cellmax, Lanzhou, China) at room temperature for 1 h and incubated with a mouse anti-human CD44 monoclonal antibody (Cat. #3570; Cell Signaling Technology, Danvers, MA, USA) and a rabbit anti-human CD24 antibody (Cat. #ab202073; Abcam, Cambridge, MA, USA) at 4ºC overnight. After being washed with phosphate-buffered saline (PBS), the sections were incubated with Alexa Fluor 647-labeled rabbit goat anti-mouse IgG and Alexa Fluor 488-labeled goat anti-rabbit IgG, followed by nuclear staining with 4',6-diamidino-2-phenylindole (DAPI). The fluorescence signals in the immunostained tissue sections were photoimaged under a fluorescence microscope (E800, Nikon, Tokyo, Japan) with the NIS-Elements F3.0 (Nikon) and analyzed using ImageJ software (National Institute of Heath, Bethesda, MD, USA). The percentages of CD44+/CD24- CSCs and CD44-/CD24- cells in 2000 tumor cells from at least three sections were calculated in a blinded manner. The tumor cells were identified, based on hematoxylin and eosin (H&E) staining in their consecutive sections (see below).

The consecutive tissue sections were deparaffinized, rehydrated and routine-stained with H&E solution. They were photoimaged and the numbers of tumor cells in each section were quantified by two pathologists in a blinded manner.

Cell lines and culture

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Human TNBC MDA-MB-231 and MDA-MB-468 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). The identity of these cell lines was confirmed by STR and the cells were tested negative for mycoplasma contamination throughout the experimental period. The cells were maintained in Leibovitz’s L15 medium (Thermo Fisher, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin (as the complete L15 medium) in a humidified incubator without addition of CO2 at 37ºC.

The sorted CD44-/CD24- cells (see below) were cultured in the complete L15 medium or the stem cell (SC) medium (10% human MammoCult Proliferation Supplements in MammoCult Basal Medium, Stem Cell Technologies, San Diego, CA, USA) for 7 days. The cells were subjected to different assays (see below).

Cell transfection

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The purified CD44-/CD24- MDA-MB-231 cells (5 × 105 cells/well) were cultured in 6-well plates overnight and transfected with the RHBDL2-specific siRNA (5'-CAUACUUGGAGAGAGAGCUAATT-3'), or negative control scramble siRNA (5'-UUCUCCGAACGUGUCACGUTT-3') from GenePharma (Shanghai, China) using Mission siRNA transfection reagents (Sigma-Aldrich, St. Louis, MO, USA) for 48 h. The efficacy of specific gene silencing was evaluated by Western blot analysis.

Flow cytometry

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Parental MDA-MB-231 (wild-type, WT), RHBDL2-silenced MDA-MB-231, MDA-MB-468/Ctrl, and MDA-MB-468/si cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD24 (Cat. #311104; BioLegend, San Diego, CA, USA) and PE-conjugated anti-CD44 (Cat. #338808; BioLegend) antibodies. Control cells were stained with an isotype control, FITC-anti-CD24 or PE-anti-CD44 alone. Subsequently, the percentages of CD44-/CD24-, CD44-/CD24+, CD44+/CD24+, and CD44+/CD24- cells were analyzed by flow cytometry in a FACS Aria III flow cytometer (BD Biosciences, San Jose, CA, USA). The same protocol was used for analyze the CD44-/CD24- cells after culture for 7 days and freshly prepared xenograft tumor cells.

Western blotting

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The different groups of cells were lyzed in radioimmunoprecipitation (RIPA) lysis buffer containing phenylmethane sulfonyl fluoride (PMSF), protease and phosphatase inhibitors and centrifuged. The cell lysate supernatants were collected, and the total protein concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo-Fisher), according to the manufacturer’s instructions. Next, the cell lysate samples (50 µg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% nonfat dry milk in Tris-based saline-Tween 20 (TBS-T) and probed overnight at 4°C with various primary antibodies (Supplementary file 4). The bound antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000 dilution; Cat. #ZDR-5306, ZDR-5307, or ZSGB-BIO, Beijing, China), and the immunoblotting signals were visualized using enhanced chemiluminescence reagents (Cat. #34076; Thermo-Fisher, Waltham, MA, USA) on a chemiluminescence instrument C300 (Azure, Dublin, CA, USA). The relative levels of individual targeted proteins to control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by densitometric analysis using ImageJ software.

RNA sequencing (RNA-seq) analysis of single cells

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Human OCT4 promoter region (1-2012 bps) was amplified by polymerase chain reaction (PCR). The generated DNA fragment, together a DNA fragment for encoding the enhanced green fluorescent protein (EGFP), was cloned into the plasmid of pGL3-basic to generate a pGL3-OCT4-EGFP plasmid for the OCT4 promoter–controlled EGFP expression. The plasmid was sequenced. Next, the purified CD44-/CD24- MDA-MB-231 cells were transfected with pGL3-OCT4-EGFP using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) and 6 h later, the cells were cultured into microraft QuAscount assay plates (Teacon, Maennedorf, Switzerland) in a single cell manner for 1, 3 or 5 days, according to the manufacturer’s protocol. The EGFP- cells at 24 h post culture and EGFP+ cells at 72 and 120 h post culture (three cells per time point) were captured separately and subjected to RNA-seq analysis at a single cell level to identify differentially expressed genes (DEGs). In brief, total RNA was isolated from individual single-cell samples, and their mRNA was enriched using the oligo-dT microbeads and fragmented to 300-500 nucleosides, followed by reversely transcribed into cDNA. The cDNA samples were amplified by PCR to generate cDNA libraries, which were sequenced in an Illumina HiSeq (Illumina, San Diego, CA, USA). The high-quality reads were aligned to the mouse reference genome (GRCm38) using the Bowtie2 v2.4.2 (Baltimore, MD, USA), and the expression levels of individual genes were normalized to the fragments per kilobase of the exon model per million mapped reads from RNA-seq by expectation maximization. The DEGs were considered if the gene expression level had a fold change of >2 and had an adjusted p-value of <0.05 between two time points.

Bioinformatics

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The DEGs were further analyzed by gene ontology (GO) using the online tool AmiGO (http://www.geneontology.org) and Database for Annotation, Visualization and Integrated Discovery (Burstein et al.) software. The potential pathways the DEGs involved were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg) annotations.

A mouse model of xenograft tumor and lung metastasis assays

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The experimental protocol was approved by the Animal Research and Care Committee of China Medical University (Shenyang, China; Project identification code: 2018PS312K, date on 03/05/2018), according to the Guidelines of the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research. Female BALB/c nude mice (6 weeks old) were obtained from Human Silaikejingda Laboratory Animals (Changsha, China) and housed in a specific pathogen-free facility with free access to autoclaved food and water. The purified CSCs from parental MDA-MB-231 and CD44-/CD24- converted MDA-MB-231 CSCs (2 × 103 cells/mouse) were injected subcutaneously into individual BALB/c nude mice. Their tumor growth and body weights were monitored up to 21 days post-cancer cell inoculation. At the end of the experiment, subcutaneous tumors were dissected and weighed. In addition, some tumor xenografts were dissected from each group of mice at 7 and 21 days post inoculation and digested to prepare single-cell suspensions for staining with FITC-anti-CD24, PE-anti-CD44, or isotype controls for flow cytometric sorting of CD44+/CD24-, CD44-/CD24-, CD44-/CD24+, and CD44+/CD24+ cells.

Moreover, the sorted CD44+/CD24- CSCs (1 × 103 cells/mouse) from parent MDA-MB-231 cells and CD44-/CD24- converted MDA-MB-231 cells were injected into the tail vein of NOD/SCID mice and on 21 days post inoculation, the mice were euthanized and their lungs were dissected and weighed (including bronchi). Additionally, the dissected lung tissue sections were stained with H and E and photographed (n=5–8 per group). The sizes of metastatic breast cancer nodules in individual mice were measured in a blinded manner.

Statistical analysis

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The data are expressed as the mean ± standard deviation (SD) from at least three separate experiments, and the difference between the groups was analyzed by Chi-Square test, Student’s t tests, or Mann–Whitney U test where applicable. The DFS and OS of each group of patients were estimated by the Kaplan-Meier method and analyzed by the log-rank test. All statistical analyses were performed using SPSS 23.0 (SPSS, Inc, Chicago, IL, USA). A p-value of ≤ 0.05 was considered statistically significant.

Results

Association of a higher frequency of CD44-/CD24- tumor cells with delayed distant metastasis in human breast cancer patients

CD44+/CD24- breast CSCs are crucial for the prognosis of breast cancer (Kaverina et al., 2017; Mylona et al., 2008), but the potential prognostic values of CD44-/CD24- breast cancer cells are rarely studied. Based on H and E staining for identification of tumor cells, immunofluorescence was used to quantify the frequency of CD44-/CD24-, CD44+/CD24-, CD44-/CD24+, and CD44+/CD24+ tumor cells in 576 breast cancer tissue specimens, including a training group (n = 355) and testing group (n = 221) using anti-CD44 and anti-CD24 antibodies. Their demographic and clinical characteristics are summarized in Supplementary file 1 and representatively histological and immunofluorescent images are shown in Figure 1—figure supplement 1. The arrows are separately indicating CD44+/CD24- cells and CD44-/CD24- cells in Figure 1—figure supplement 1A and B. There were varying percentages of CD44-/CD24-, CD44+/CD24-, CD44-/CD24+, and CD44+/CD24+ tumor cells in Figure 1—figure supplement 2A. After stratification of the training group of patients, based on their distant metastasis, the frequency of CD44-/CD24- cells in breast cancer tissues of patients with postoperative distant metastasis was significantly higher than those without distant metastasis (p<0.0001; Figure 1A). The average frequency of CD44-/CD24- cancer cells in all samples was 19.7% with a median value of 19.5%. The ROC analysis revealed a cut-off value of 19.5% with a sensitivity of 70.5%, specificity of 71.2% and Youden index of 0.471 (Figure 1B; Supplementary file 2). Accordingly, the patients in the training set were stratified into two subgroups with high or low frequency of CD44-/CD24- cells. In the training set of patients, the metastasis rate in the patients with a high frequency (>19.5%) of CD44-/CD24- breast cancer cells was 1.97-fold higher than those with a low frequency (<19.5%) of CD44-/CD24- tumor cells. Analysis of different molecular subtypes of breast cancers indicated that the metastatic rates in the patients with a high frequency of CD44-/CD24- breast cancer cells were significantly higher than those with a low frequency of this type of cells (i.e. 40.85% vs. 6.82% for luminal; 47.06% vs. 13.64% for HER-2+; 63.79% vs. 22.22% for TNBC with a high vs. low frequency of CD44-/CD24- tumor cells; Figure 1C). Univariate and multivariate analyses indicated that high frequency of CD44-/CD24- cancer cells or CSCs, positive lymph node metastasis, higher N stage, and TNBC were independent risk factors for poor DFS (Supplementary file 3).

Figure 1 with 3 supplements see all
Association of a high frequency of CD44-/CD24- cells in breast cancer tissues with delayed tumor distant metastasis.

(A) Immunofluorescent analysis of the percentages of CD44-/CD24- cells in tissue samples from breast cancer patients with or without tumor metastasis. The P-value was determined by Student’s t-test. N = 105 and 250 for with and without metastasis, respectively. (B) ROC analysis of the sensitivity and specificity of 19.5% of CD44-/CD24- cells as a cutoff value for evaluating delayed distant metastasis. (C) Metastasis rates in patients with different molecular subtypes of breast cancer after they were stratified into high (≥19.5%) or low frequency of CD44-/CD24- cells. (D) Metastasis rates in patients with ≥2% CD44+/CD24- cells (n = 105) vs. C1 patients with <2% CD44+/CD24- cells and ≥19.5% CD44-/CD24- cells (n = 69). (E) Kaplan–Meier analysis of the DFS of breast cancer patients after they were stratified, based on the indicated measure. (F) Kaplan–Meier analysis of the DFS of TNBC patients with standard chemotherapy after they were stratified into ≥19.5% CD44-/CD24- cells (n = 52); <19.5% CD44-/CD24- cells (n = 80). (G) Kaplan–Meier analysis of the DFS of luminal breast cancer patients with standard endocrine therapy after they were stratified into ≥19.5% CD44-/CD24- cells (n = 24) vs. <19.5% CD44-/CD24- cells (n = 23).

Furthermore, analysis of metastatic dynamics revealed that patients with a higher frequency (≥2%) of CD44+/CD24- CSCs usually developed distant metastasis earlier than those with < 2% of CD44+/CD24- and >19.5% of CD44-/CD24- tumor cells (the C1 group, Figure 1D). Similarly, the DFS of patients with a high frequency of CD44-/CD24- tumor cells or CD44+/CD24- CSCs was significantly shorter than those with a low frequency of corresponding cells (Figure 1E). The similar patterns of DFS were observed in three subtypes of breast cancer patients (Figure 1—figure supplement 2D and Figure 1—figure supplement 3A). The metastatic dynamics also indicated that patients with a high frequency of CSCs had a metastatic peak near 5 years after surgical resection of the tumor (Figure 1—figure supplement 2C). Some patients with a higher frequency of CD44-/CD24- tumor cells and CSCs had the highest rate of postoperative metastasis in this population. However, to exclude the effects of CSCs, patients in the C1 group also had a higher risk to develop delayed distant metastasis during 5–7 years post tumor resection than those with <2% of CD44+/CD24- CSCs and < 19.5% of CD44-/CD24- tumor cells (the C0 group, Figure 1E). Because postoperative therapies also affect the DFS, we further analyzed the frequency of CD44-/CD24- cells in TNBC patients with chemotherapy and luminal breast cancer patients with hormone therapy. As shown in Figure 1F and G, TNBC and luminal breast cancer patients with a higher frequency of CD44-/CD24- cells had a worse DFS, compared than those with a lower frequency of CD44-/CD24- cells following standard therapies. Hence, a higher frequency of CD44-/CD24- breast cancer cells, like higher frequency of CD44+/CD24- CSCs, was associated significantly with a shorter DFS in the training set of patients regardless of standard therapies.

The frequency of CD44-/CD24- cells predicts the delayed distant metastasis in breast cancer patients following standard postoperative treatment

To validate the importance of CD44-/CD24- cell frequency in delayed metastasis, the testing set of patients was analyzed and the metastatic rates of patients with a higher frequency of CD44-/CD24- cells were higher than those with a lower frequency of CD44-/CD24- cells in the tested molecular subtypes of breast cancers (22.73% vs. 8.89% for luminal; 50% vs. 18.42% for HER-2+; 41.67% vs. 15% for TNBC with high vs. low CD44-/CD24- cells; Figure 2A). The DFS and OS of patients with a higher frequency of CD44-/CD24- cells were significantly shorter than those with a lower frequency of CD44-/CD24- cell cells in the testing set (Figure 2B,C). Similarly, the DFS and OS of patients with a higher frequency of CD44-/CD24- cells were significantly shorter than those with a lower frequency of them, regardless of their molecular subtypes (Figure 2—figure supplement 1A,B). The metastatic dynamics revealed that while higher and earlier metastatic rates were observed in the testing set of patients, particularly for those with a high frequency of CD44+/CD24- CSCs and those with < 2% of CD44+/CD24- CSCs and >19.5% of CD44-/CD24- tumor cells in the (C1 group) developed delayed distant metastasis, which peaked between 4 and 5 years post tumor resection (Figure 2D).

Figure 2 with 1 supplement see all
The higher frequency of CD44-/CD24- tumor cells is associated with delayed distant metastasis and worse DFS in the testing group of patients.

(A) Postoperative metastasis rate in the test group of patients with different molecular subtypes of breast cancer after they were stratified by 19.5% of CD44-/CD24- cells. (B and C) Kaplan–Meier analysis of the DFS (B) and OS (C) of all patients in the testing group after they were stratified into ≥19.5% of CD44-/CD24- (n = 100) vs. <19.5% of CD44-/CD24- tumor cells (n = 121). (D) Longitudinal measurements of metastasis rates among all breast cancer patients (n = 211), patients with ≥2% CD44+/CD24- cells (n = 68) and the C1 group of patients with <2% of CD44+/CD24- and ≥19.5% CD44-/CD24- cells (n = 153). (E) Kaplan–Meier analysis of the DFS in the testing group of TNBC patients with chemotherapy after they were stratified into ≥19.5% of CD44-/CD24- (n = 24) vs. <19.5% of CD44-/CD24- tumor cells (n = 35). (F) Kaplan–Meier analysis of the DFS in the testing group of luminal breast cancer patients with endocrine therapy after they were stratified into ≥19.5% of CD44-/CD24- (n = 22) vs. <19.5% of CD44-/CD24- tumor cells (n = 19). p-Values were determined by log-rank test.

Furthermore, following standard chemotherapy or endocrine therapy, the patients with a higher frequency of CD44-/CD24- tumor cells had a significantly shorter DFS than their corresponding patients with a lower frequency of CD44-/CD24- tumor cells (Figure 2E,F). Similar results were achieved in the training set of patients following chemotherapy and the patients with a higher frequency of CD44-/CD24- cells had a higher risk to develop distant metastasis beginning at 4 years post tumor resection (Figure 1—figure supplement 3B). Moreover, patients in the C1 group, with a low frequency of CSCs and higher frequency of CD44-/CD24- tumor cells, also had a worse DFS than those with a lower frequency of CD44-/CD24- tumor cells in the C0 group (Figure 1—figure supplement 3C). A similar pattern of DFS was observed in TNBC patients and those with a higher frequency of CD44-/CD24- cells had a high risk to develop progressive metastasis at 4 years post tumor resection regardless of chemotherapy (Figure 2E). In this regard, the frequency of CD44-/CD24- cells in tumor tissues could be a valuable predictor of standard therapeutic response in breast cancer patients. Similar data were observed in the testing group of patients (Figure 2E and Figure 2—figure supplement 1C,D). Thus, a higher frequency of CD44-/CD24- tumor cells was associated with worse survival of breast cancer patients following standard therapies.

Spontaneous conversion of CD44-/CD24- TNBC cells into CD44+/CD24- CSCs in vitro and in vivo

A previous study has shown that the differentiated breast cancer cells can spontaneously convert into CSCs to renew the CSC pool, although it remains unclear whether the CSCs derived from the differentiated breast cancer cells have the same biological behaviors as the original CSCs (Najafi et al., 2019). Because breast CSCs are crucial for the metastasis of breast cancer, whether CD44-/CD24- cells could convert into CSCs was tested in vitro and in vivo. First, primary human tumor cells were isolated from TNBC breast cancer patients and primary CD44+/CD24- CSCs and CD44-/CD24- tumor cells were purified by flow cytometry sorting (Figure 3A, Figure 3—figure supplement 1A). Subsequently, CD44-/CD24- cells were cultured for 7 days. There were 4.6% of CD44+/CD24- CSCs (Figure 3B). Following implantation with CD44-/CD24- MDA-MB-231 cells in the breast fat pad of female BALB/c nude mice, 10.8–16.3% of CD44+/CD24- CSCs were detected in the formed tumors at 7 and 21 days post implantation in mice, respectively (Figure 3C). Clearly, CD44-/CD24- TNBC cells effectively converted into CD44+/CD24- CSCs in vitro and in vivo. Because TNBC is the most aggressive type of breast cancer, the spontaneous conversion of CD44-/CD24- TNBC cells into CD44+/CD24- CSCs was further tested in TNBC cell lines (Figure 3D and Figure 3—figure supplement 1). CD44+/CD24- parent CSCs and CD44-/CD24- cells were purified from MDA-MB-231 and MDA-MB-468 cells by flow cytometry sorting and CD44-/CD24- cells were cultured in both SC and L15 media for 7 days, respectively. Following culture of CD44-/CD24- cells from MDA-MB-231 cells, flow cytometry analysis exhibited 28.3% and 24.7% of CD44+/CD24- cells in SC and L15 media, respectively (Figure 3E). Similar results were observed for the parental MDA-MB-468 cells (Figure 3—figure supplement 1B).

Figure 3 with 1 supplement see all
Spontaneous conversion of CD44-/CD24- TNBC cells into CD44+/CD24- CSCs.

(A) A diagram Illustrated the experimental protocol for testing the spontaneous conversion of primary human breast cancer CD44-/CD24- cells into CD44+/CD24- CSCs in vitro. (B, C) Flow cytometry analysis of the spontaneous conversion of primary human or xenograft breast cancer CD44-/CD24- cells into CD44+/CD24- CSCs in vitro. The primary human and xenograft breast cancer CD44-/CD24- cells were purified from human fresh TNBC tissue cells or MDA-MB-231 xenograft tissue cells by flow cytometry sorting and cultured in L15 medium for 7 and 21 (specifically for cells from xenograft tissue cells) days, respectively. The percentages of CD44+/CD24- CSCs were analyzed by flow cytometry. (D) A diagram illustrated the experimental protocol for testing the spontaneous conversion of CD44-/CD24- cells from TNBC cells into CD44+/CD24- CSCs. (E) Flow cytometry analysis of the percentages of CD44+/CD24- CSCs. CD44-/CD24- MDA-MB-231 cells were purified by flow cytometry sorting and cultured in the indicated medium for the indicated duration, followed by flow cytometry analysis.

Parental TNBC CSCs and newly converted CSCs from CD44-/CD24- TNBC cells display similar biological behaviors in vitro

Next, the biological behaviors of the newly converted CD44+/CD24- CSCs from CD44-/CD24- MDA-MB-231 cells (CD44-/CD24- CSCs) and parental CSCs directly purified from MDA-MB-231 cells (WT CSCs) were measured for their mammosphere formation, self-renewal, tumor cell differentiation, and CSC stemness marker expression in vitro and their tumorigenicity in vivo. Both WT CSCs and CD44-/CD24- CSCs formed similar numbers of mammospheres with comparable sizes (Figure 4A) and displayed similar proliferative capacity (Figure 4B). Culture of both types of CSCs for 7 days promoted their differentiation into different subtypes of MDA-MB-231 cells with similar percentages (Figure 4C,D). Western blot analysis revealed that the relative levels of OCT4, SOX2, NANOG, NESTIN, and ABCB1 proteins were also comparable between these two types of CSCs (Figure 4E). Thus, both types of CSCs exhibited comparable capacities to self-renew, differentiate and form mammospheres, and had similar stemness properties.

Both WT CSCs and CD44-/CD24- CSCs from TNBC cells have similar biological behaviors in vitro.

(A) CD44-/CD24- CSCs and WT CSCs displayed similar ability to form mammosphere in vitro following culturing them for 7 days. (B) CCK-8 assay analysis of WT and CD44-/CD24- CSC proliferation. (C) Flow cytometry analysis of the frequency of CSCs after culture WT and CD44-/CD24- CSCs in SC medium for 7 days. (D) Flow cytometry analysis of WT and CD44-/CD24- CSC differentiation after culturing them in SC medium for 7 days. Data are representative images or expressed as the mean or mean ± SD of each group from three independent experiments. (E) Western blot analysis of stemness marker expression in WT and CD44-/CD24- CSCs.

Both WT CSCs and CD44-/CD24- CSCs from TNBC cells have similar tumorigenesis and distant metastasis properties in vivo

Next, whether both types of CSCs functioned similarly was tested in BALB/c nude mice. After implantation with the same number of each type of CSCs for 21 days, both types of CSCs induced xenograft tumors with similar sizes and weights (Figure 5A). Flow cytometric analysis of single cells from these xenograft tumors exhibited similar percentages of different subtypes of breast cancer cells (Figure 5B). Furthermore, intravenous injection of equal number of each type of CSCs induced lung metastatic nodules with similar tumor sizes and lung weights in NOD/SCID mice (Figure 5C). H and E staining of lung tissue sections revealed similar pathological characters in both groups of mice (Figure 5C). Thus, both WT CSCs and CD44-/CD24- CSCs from TNBC cells had similar tumorigenicity and metastatic capacities in vivo.

Both WT and CD44-/CD24- CSCs from MDA-MB-231 cells exhibit similar tumorigenicity and comparable abilities to differentiate and induce lung metastatic in vivo.

(A) Both WT and CD44-/CD24- CSCs had similar tumorigenicity to induce comparable sizes of tumors in BALB/c nude mice following subcutaneous implantation for 21 days (n = 5–8 per group). (B) Flow cytometry analysis of different subtypes of TNBC cells in xenograft tumors. (C) Lung metastasis. NOD/SCID mice were intravenously injected with the same number of WT or CD44-/CD24- CSCs and 21 days later, the mice were euthanized and their lungs were dissected for weighing and histological H and E staining to examine lung metastatic morphology and nodule sizes. Data are representative images or expressed as the mean ± SD of each group (n = 5–8 per group), and samples were analyzed from three independent experiments.

RHBDL2 is crucial for spontaneous conversion of CD44-/CD24- breast cancer cells into CD44+/CD24- CSCs

To understand the molecular mechanisms underlying spontaneous conversion of TNBC CD44-/CD24- cells, CD44-/CD24- cells were first purified from MDA-MB-231 cells by flow cytometry and transfected with pGL3-OCT4-EGFP using Lipofectamine 3000. The cells were cultured into micrograft plates in a single cell manner for 1, 3, and 5 days (Figure 6A). Subsequently, the cultured individual cells were captured and subjected to RNA-seq analysis to identify DEGs.

Figure 6 with 1 supplement see all
RHBDL2 expression is up-regulated during the process of CD44-/CD24- MDA-MB-231 cell conversion into CD44+/CD24- CSCs.

(A) Representative images of micrograft plates for culture of CD44-/CD24- MDA-MB-231 cells at a single cell level. CD44-/CD24- MDA-MB-231 cells were purified by flow cytometry sorting and transfected with pGL3-OCT4-EGFP, followed by culturing them in micrograft plates at a single cell level for 24, 72, and 120 hr, and the average of the gene expression profile of three cells are photographed. (B) RNA-seq analysis and heatmap displayed the top DEGs during the process of CD44-/CD24- MDA-MB-231 cell conversion into CD44+/CD24- CSCs. (C) Kaplan-Meier estimation of the association of the expression of DEGs with DFS in breast cancer patients in TCGA database. (D) RT-qPCR analysis of the relative levels of gene mRNA transcripts in WT CD44+/CD24-, CD44-/CD24- CSCs, and unconverted CD44-/CD24- cells after culture for 7 days. Data are representative images or expressed as the mean ± SD of each group from at least three separate experiments. *p<0.05, ***p<0.001; ##p<0.01, ###Pp<0.001 vs. the CD44-/CD24- cells, determined by Student’s t-test.

There were 11 DEGs associated with tumor cell differentiation and dedifferentiation between EGFP- CD44-/CD24- cells and EGFP+ CSCs and they included RHBDL2, HIST1H4H, DSCC1, ZNF710, ATP8B3, and others. Particularly, comparison of EGFP+ CSCs (120 hr post culture) with EGFP- (24 hr post culture) and uncommitted cells (72 hr post culture) revealed that both RHBDL2 and HIST1H4H mRNA transcripts significantly increased (Figure 6B). GO and KEGG analyses revealed that all DEGs were predominantly involved in cell organelle formation, metabolism, signal transduction, and transcriptional regulation (Figure 6—figure supplement 1A,B). Furthermore, Kaplan-Meier analysis and log-rank test indicated that higher levels of HIST1H4H, RHBDL2, DSCC1, ARL6IP1, PPME1, and lower levels of G2E3 and MED22 mRNA transcripts, but not others, were significantly associated with worse DFS in breast cancer patients in the Cancer Genome Atlas (TCGA) database (Figure 6C). Finally, the levels of mRNA transcripts of these DEGs among the purified parental CD44+/CD24- CSCs (red color), the newly converted CD44+/CD24- CSCs (blue color) and the unconverted CD44-/CD24- tumor cells (green color) from MDA-MB-231 cells were tested by RT-qPCR. Compared with the unconverted CD44-/CD24- tumor cells, PPME1, RHBDL2 and HIST1H4H mRNA transcripts increased in the newly converted CD44+/CD24- CSCs with a change of >two folds, which were similar to that in parental CD44+/CD24- CSCs (Figure 6D). Together, these data suggest that up-regulated expression of these genes may be crucial for the spontaneous conversion of CD44-/CD24- TNBC cells into CD44+/CD24- CSCs. Given that RHBDL2 mRNA transcripts at 120 hr post culture were the highest among the different time points post culture, the following experiments centered on the role of RHBDL2 in the spontaneous conversion of CD44-/CD24- TNBC into CD44+/CD24- CSCs and their malignant behaviors.

RHBDL2 silencing inhibits the YAP1/UPS31/nuclear factor (NF)-κB signaling and spontaneous CD44-/CD24- cell conversion into CD44+/CD24- CSCs

Next, how RHBDL2 and the related signaling affected in the spontaneous CD44-/CD24- cell conversion into CD44+/CD24- CSCs was explored by silencing RHBDL2 expression in TNBC cells using siRNA-based technology. Because the YAP1 signaling can suppress USP31 expression, a potent inhibitor of the NF-κB signaling, how RHBDL2 silencing could affect the relative levels of YAP1 expression and phosphorylation was investigated in MDA-MB-231 and MDA-MB-468 cells. Compared with the controls, RHBDL2 silencing dramatically decreased RHBDL2 and YAP1 expression and slightly increased YAP1 phosphorylation in both TNBC cells (Figure 7A). Furthermore, RHBDL2 silencing also obviously increased the relative levels of USP31 expression, YAP1 phosphorylation and decreased the relative levels of NF-kB phosphorylation, besides the demolished RHBDL2 expression, in CD44-/CD24- cells sorted from both the negative control (NC) and RHBDL2-silenced MDA-MB-231 cells and MDA-MB-468 cells (Figure 7B). Moreover, RHBDL2 silencing not only decreased YAP1 expression in the cytoplasm, but also dramatically reduced the levels of nuclear YAP1 in both TNBC cells (Figure 7C). In addition, after culture of control CD44-/CD24- and RHBDL2-silenced CD44-/CD24- cells in L15 medium for 7 days, the results indicated that RHBDL2 silencing remarkably decreased the percentages of CD44+/CD24- CSCs from 6.5–5.5% to 0.5–0.7% in both types of TNBC cells (Figure 7D,E). Finally, we determined if RHBDL2 silencing could modulate the mammosphere formation of CD44+/CD24- CSCs in vitro. CD44-/CD24- cells were purified from MDA-MB-231 and MDA-MB-468 cells and transfected with control siRNA or RHBDL2-specific siRNA, followed by culturing them in L15 medium for 7 days. Subsequently, their ability to form mammospheres was examined. Compared with the control siRNA-transfected cells, RHBDL2 silencing significantly decreased the numbers and sizes of formed mammospheres in both types of TNBC cells (Figure 7F). Therefore, these findings further suggest that RHBDL2 may be a crucial regulator of the spontaneous conversion of CD44-/CD24- TNBC cells into CD44+/CD24- CSCs by enhancing the YAP1/USP31/NF-κB signaling in breast cancer cells.

RHBDL2 silencing inhibits the spontaneous conversion of CD44-/CD24- cells into CSCs by attenuating the YAP1/NF-kB signaling through enhancing USP31 expression in TNBC cells.

(A) Western blot analysis exhibited that RHBDL2 silencing decreased RHBDL2 and YAP1 expression, but increased YAP1 phosphorylation in TNBC cells. (B) Western blot analysis displayed that RHBDL2 silencing decreased RHBDL2 and YAP1 expression, NF-kB activation, but increased USP31 expression and YAP1 phosphorylation in the indicated TNBC CD44-/CD24- cells. (C) Western blot analysis of cytoplasmic and nuclear YAP1 protein levels in MDA-MB-231/MDA-MB-468 cells following RHBDL2 silencing indicated that RHBDL2 silencing mitigated the nuclear translocation of YAP1 in TNBC cells. (D and E) RHBDL2 silencing inhibited the spontaneous conversion of CD44-/CD24- cells into CSCs in vitro. CD44-/CD24- cells were purified from MDA-MB-231 (D and E) RHBDL2 silencing inhibited the spontaneous conversion of CD44-/CD24- cells into CSCs in vitro. CD44-/CD24- cells were purified from MDA-MB-231 (D) and MDA-MB-468 (E ) cells and transfected with the control or RHBDL2-specific siRNA, followed by culturing them in L15 medium for 7 days. Subsequently, the percentages of CD44+/CD24- CSCs in each group of cells were analyzed by flow cytometry. (F) RHBDL2 silencing attenuated mammosphere formation of CD44-/CD24- CSCs. Following transfected with the control or RHBDL2-specific siRNA, CD44-/CD24- CSCs were cultured in L15 medium for 7 days and the formed mammospheres were photoimaged (magnification x 400) and their numbers and sizes were measured in a blinded manner. Data are representative images or expressed as the mean ± SD of each group from at least three separate experiments. *p <0.05, **p <0.01, determined by Student t-test. (G) A diagram illustrates the possible mechanisms underlying the action of RHBDL2 in enhancing the spontaneous conversion of CD44-/CD24- cells into CD44+/CD24- CSCs, contributing to delayed distant metastasis of breast cancer.

Discussion

The current study uncovered that CD44-/CD24- TNBC cells were able to spontaneously convert to CD44+/CD24- CSCs, consistent with previous studies in vitro (Italiano and Shivdasani, 2003; Zoppino et al., 2010) both WT CSCs and CD44-/CD24- CSCs had similar biological properties in terms of stemness marker expression, self-renewal, differentiation, tumorigenicity and lung metastasis in vitro and in vivo a higher frequency (≥19.5%) of CD44-/CD24- cells in breast cancer tissues was associated with worse DFS and delayed postoperative distant metastasis a higher frequency of CD44-/CD24- and CD44+/CD24- cells, higher tumor N stage, and molecular subtypes were predictors of DFS for breast cancer patients; and mechanistically, RHBDL2 silencing inhibited the YAP/USP31/NF-κB signaling and attenuated the spontaneous CD44-/CD24- cell conversion into CD44+/CD24- CSCs and their mammosphere formation. Therefore, the findings may uncover new biomarkers for prognosis of delayed distant breast cancer metastasis and shed light on the molecular mechanisms underlying the distant metastasis of breast cancer, particularly for TNBC. It is necessary to further investigate whether RHBDL2 can be a therapeutic target in inhibiting the development and progression of breast cancer.

Previous studies have shown that non-CSC tumor cells can dedifferentiate (convert) into CSCs in human glioblastoma and intestinal stroma melanoma, contributing to intra- and inter-tumor heterogeneity (Stepanova et al., 2003; Tzimas et al., 2006; Wei et al., 2019). Furthermore, CSC plasticity and heterogeneity can promote tumor progression and resistance to therapy (Das et al., 2020; Kilmister et al., 2020; Martin-Castillo et al., 2013; Thankamony et al., 2020). For example, intra-tumoral heterogeneity is a major ongoing challenge for effective cancer therapy, while CSCs are responsible for intra-tumoral heterogeneity, therapeutic resistance, and metastasis, which may be because cancer cells exhibit a high level of plasticity and an ability to dynamically switch between CSC and non-CSC states or among different subsets of CSCs (Thankamony et al., 2020). Consistently, the differentiated non-CSCs can revert to be trastuzumab-refractory, CSS-like cells by enhancing their epithelial-to-mesenchymal transition process (Martin-Castillo et al., 2013). Similarly, the changes in tumor microenvironment and epigenetics can promote the conversion of non-CSCs into CSCs, leading to tumor progression and therapeutic resistance (Das et al., 2020). The current study further confirmed that CD44-/CD24- TNBC cells spontaneously converted into CD44+/CD24- CSCs that possessed biological properties, similar to their CSCs isolated directly from parental TNBC cells. More importantly, a higher frequency of CD44-/CD24- cells in breast cancer tissues was associated with significantly with worse DFS and delayed distant metastasis. These, together with the dynamic process of spontaneous conversion of non-CSC CD44-/CD24- cells and CSC differentiation, indicate that the spontaneous conversion of CD44-/CD24- cells helps maintain the CSC pool size, contributing to the delayed distant metastasis and worse prognosis in breast cancer patients.

The data from the current study indicated that a higher frequency of CD44-/CD24- cancer cells, like higher frequency of CSCs, was one of the independent risk factors for delayed distant metastasis and worse DFS in this population. Particularly, the higher frequency of CD44-/CD24- cancer cells was a better predictor of delayed breast cancer metastasis (up to 12 years after initial breast cancer diagnosis). Evidently, while high frequency of CSCs was valuable for predicting distant metastasis at 5–7 years post-surgery a higher frequency of CD44-/CD24- cells effectively predicted delayed distant metastasis in cases with a low frequency of CSCs. These results suggest that CD44-/CD24- cells may spontaneously convert into CSCs and cause delayed distant metastasis when environmental and other factors cause a clonal evolution by accumulating successive mutations (Meacham and Morrison, 2013).

In addition, the RNA-seq analysis and subsequent RT-qPCR identified several DEGs during the dynamic process of CD44-/CD24- TNBC cell conversion into CSCs, such as RHBDL2, and its expression was up-regulated in the newly converted CSCs. The RHBDL2, also known as the rhomboid like 2, is one intramembrane serine protease of the secretase-A rhomboid family (Etheridge et al., 2013). Previous studies have identified that the RHBDL2 cleaves its substrates, including EGF, ephrin-β3, thrombomodulin, the C-type lectin family 14 member A (CLEC14A), cadherin, IL-6R, Spint-1 and the collagen receptor tyrosine kinase DDR1 (Adrain et al., 2011; Battistini et al., 2019; Etheridge et al., 2013). Although the functional consequence of cleaving these substrates has not been fully explored the available findings indicate that RHBDL2 functionally activates the EGF signaling, cadherin shedding, angiogenesis and promotes the wound healing and migration of different types of cells, including tumor cells (Adrain et al., 2011; Cheng et al., 2011). Furthermore, up-regulated RHBDL2 expression is associated with worse prognosis of several types of malignant tumors, such as breast cancer, pancreatic adenocarcinoma, clear cell kidney cancer, and low-grade glioma patient (Canzoneri et al., 2014; Johnson et al., 2017). However, little is known on how upregulated RHBDL2 expression promotes malignant behaviors of these types of tumors. Interestingly, the current study not only observed that the upregulated RHBDL2 transcription was associated with worse DFS of breast cancer patients in TCGA, but also found that RHBDL2 silencing inhibited the YAP1/NF-κB signaling by upregulating USP31 expression and attenuated the spontaneous conversion of CD44-/CD24- cells into CD44+/CD24- CSCs and their mammosphere formation. Moreover, these data extended previous observation that YAP1-mediated suppression of USP31 enhances NF-κB activity to promote sarcomagenesis (Kemeny and Fisher, 2018; Mehta et al., 2018). YAP1 can enhance CSC stemness in several types of human cancers, and aberrant YAP1 activation is associated with a low level of TNBC differentiation and poor survival of breast cancer patients (Bora-Singhal et al., 2015; Hansen et al., 2015). Therefore, the findings from the current study indicated that RHBDL2 promoted the spontaneous conversion of CD44-/CD24- cells into CD44+/CD24- CSCs by enhancing YAP1 stability through inhibiting its phosphorylation to suppress USP31 expression, enhancing the NF-κB signaling in breast cancer cells, whereas RHBDL2 silencing had the opposite effects in breast cancer cells (Figure 7G). Alternatively, RHBDL2 may cleave other substrates to activate the EGF receptor-mediated signaling, and modify cadherin and other molecules through their involved signal pathways to promote the spontaneous conversion of CD44-/CD24- cells into CD44+/CD24- CSCs. Hence, these findings may provide new insights into the molecular mechanisms underlying the action of RHBDL2 in regulating the CSC pool to promote the metastasis of TNBC. Given that CSCs are crucial for breast cancer progression and metastasis, RHBDL2 may be a new therapeutic target for control of CD44-/CD24- cell conversion into CSCs to reduce CSC pool size and limit breast cancer progression and metastasis.

Definitely, this study had limitations. First, the sample size in some groups remained relatively smaller, which might affect statistical power. Second, this study only centered on the role of RHBDL2, but not other DEGs in regulating the spontaneous conversion of CD44-/CD24- TNBC cells into CD44+/CD24- CSCs. The results might miss some important information on the molecular mechanisms underlying the spontaneous conversion. Furthermore, the method for identification of CD44-/CD24- TNBC cells did not include a positive tumor molecular marker, which might contaminate some other types of cells, although using histological tissue sections for identification of tumor cells. Thus, further studies are warranted with advanced cutting-edge technologies, such as combination of single-cell RNA-seq, multiple marker-based CyTOF mass cytometry or spatial proteomics to quantify the frequency or number of CD44-/CD24- breast cancer cells in fresh breast cancer tissues, together with prospectively following-up the patients, in a bigger population to investigate the prognostic value of CD44-/CD24- breast cancer cells and the molecular mechanisms underlying the spontaneous conversion of CD44-/CD24- TNBC into CD44+/CD24- CSCs in the metastasis of breast cancer.

In summary, postoperative breast cancer metastasis, especially delayed metastasis (e.g. ≥5 years after diagnosis), is an important unresolved issue. The results from the current study indicated that patients with a higher frequency of CD44-/CD24- cells in their breast cancer tissues had a high risk to develop delayed distant metastasis 5–7 years after diagnosis. Mechanistically, CD44-/CD24- cells spontaneously converted into CD44+/CD24- CSCs and both WT and newly converted CSCs had similar stemness properties in vitro and in vivo. RHBDL2 silencing significantly decreased YAP1 expression and increased USP31 expression to attenuate the NF-kB activation and CD44-/CD24- cell conversion into CSCs as well as their mammosphere formation. RHBDL2 may act as a positive regulator of the spontaneous conversion of CD44-/CD24- cells into CSCs by enhancing the YAP1/USP31/NF-kB signaling. Alternatively, RHBDL2 may cleave other substrates to activate the EGF and other signal pathways, enhancing the spontaneous conversion of CD44-/CD24- TNBC into CSCs and their metastasis. These novel findings may provide insights into the molecular process of non-CSCs conversion into CSCs, leading to breast cancer progression and delayed distant metastasis. Therefore, the current findings may uncover a novel biomarker for prognosis and therapeutic target for intervention of breast cancer, particularly for TNBC.

Data availability

All data generated or analyzed during this study is included in the manuscript and supporting documents.

References

    1. Abraham BK
    2. Fritz P
    3. McClellan M
    4. Hauptvogel P
    5. Athelogou M
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    Prevalence of CD44+/CD24-/low cells in breast Cancer may not be associated with clinical outcome but may favor distant metastasis
    Clinical Cancer Research 11:1154–1159.

Decision letter

  1. Renata Pasqualini
    Reviewing Editor; Rutgers University, United States
  2. Mone Zaidi
    Senior Editor; Icahn School of Medicine at Mount Sinai, United States
  3. Robin Anderson
    Reviewer; Olivia Newton-John Cancer Research Institute, Australia

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

The manuscript describes a possible link between the presence of CD44-/CD24- cells and post-operative site distant metastasis. The functional significance of CSC, particularly of the phenotype CD44-/CD24- remains elusive and is worthy of careful evaluation. The strength of the study is the inclusion of a large number of patient samples, which were used in a retrospective analysis. The resulting data provide the rationale for the application of contemporary technologies in the evaluation of CDX and PDX models. As such, this work paves the way for further investigation at a mechanistic and translational level, which may lead to a better understanding of breast cancer metastasis.

Decision letter after peer review:

Thank you for submitting your article "Association of CD44 -/CD24 -Breast Cancer Cells with Late Stage Tumor Recurrence" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Mone Zaidi as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Robin Anderson (Reviewer #1).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. Essential revisions:

1) Referees comments must be addressed in a convincing manner, with additional data.

2) Edits are required in all sections of the manuscript.

3) If extensively revised, this manuscript would represent an incremental contribution in the field of breast cancer biology.

Reviewer #1:

The authors set out to demonstrate the importance of a subset of cancer cells lacking CD44 and CD24, in promoting tumor progression, metastasis and resistance to therapy. They analyse archival breast tissues from patients where the clinical outcome – development of metastasis and therapy response – are known. They then take this information into cell culture experiments, seeking the mechanism that underlies the importance of these CD44-/CD24- cells. Their major premise is that these double negative cells can de-differentiate into cancer stem cells that subsequently promote metastasis and chemotherapy resistance.

The overall concept of this study is good and the cell-based results are mainly clear. However, the archival tissue analysis needs clarification, as does the RNAseq analysis, as detailed below.

The breast cancer tissues are scored by immunofluorescence for expression of CD44 and CD24, but there is no indication that these two markers are being scored only in tumor cells rather than in all lineages within the tumor microenvironment. This is an important point that must be clarified. Also, the RNAseq analysis is reported to be conducted on single cells, but the data presented do not indicate if this is actually the case and how many replicate samples were analysed and which sample yielded each heat map pattern. Again clarification is required before the data can be interpreted.

Finally, the conclusion from this study is that RHBDL2 induces spontaneous conversion of CD44-/CD24- cells into CD44+/CD24- cells via YAP1, USP31 and decreased NFkB signaling. This may well be the case, but has not been proven by the experiments presented here. Whilst DHBDL2 does alter YAP, USP31 and NFkB, it could also be working through an alternate pathway to induce CSCs.

1. Figure 1 and S1 and S2A score the numbers of samples with the varying CD44/CD24 protein levels. For Figure S1, this is shown by immunofluorescence, but there is no indication of whether the cells being analysed are tumor cells or host cells from the microenvironment. There should be co-staining for EpCAM or CK19 to confirm that tumor cells only are being scored. I am assuming that the data shown in Figure S2A are actually from the immunostaining and not from flow cytometry analysis as stated in the figure legend? If this was a flow analysis, there is no description of the origin or processing of the samples reported in Figure 1A for flow cytometry. The legend for Figure S2A states that the data are stratifying only metastatic tumors, but in Figure 1A, apparently many of the tumors showed no metastasis. All these points need to be clarified/corrected.

2. This first section of the manuscript is poorly presented and I am struggling to follow the arguments and data being provided. Many of the figures are mis-labelled in the text and they are not presented in consecutive order as discussed in the text. For some references to data in the text where the wrong figure has been cited, I cannot even find the correct figure.

3. Possibly the way the data are presented, with some figures referring to the training set and other figures to the test set is adding to the confusion when it is not clearly stated which set is under discussion at the time.

4. Late metastasis – better phrased as delayed metastasis.

5. The sentence in the Introduction: "Previous studies have shown that CD44+/CD24- breast CSCs might be a dominant factor in relapse of triple negative breast cancer (TNBC), due to their possession of potent self-renewal and differentiation capacities to differentiate into mature CD44-/CD24-, CD44+/CD24+, and CD44-/CD24+ cancer cells (Geng et al., 2014; Wang et al., 2014)". It is not at all clear to me that the references provided support this comment. Geng et al. do not mention CD44-/CD24- cells at all, and Wang et al. do not discuss differentiation of CD44+/CD24- into the other phenotypes. Only CD44- cells are mentioned, not CD24.

6. Sentence in Introduction: "Indeed, injection of up to 1000 breast CSCs was able to generate a solid tumor mass in immunocompromised mice (Chaffer et al., 2011; Iliopoulos et al., 2011)." Please clarify. It is not obvious to me that Chaffer et al. specified how many CSCs they injected and Iliopoulos et al. showed that as few as 50 CSCs could form tumors.

7. Sentence in Introduction: "Moreover, previous studies reported that non-CSCs, such as CD44-/CD24- TNBC cells, are able to spontaneously convert into CSCs to renew the CSC pool, resulting in chemoresistance (Gruber et al., 2016; Kim et al., 2015; Ye et al., 2018)." The statement being referenced is for non-CSCs in TNBC, but Gruber et al. and Ye et al. are not reporting on TNBC.

8. Figure 1B – please explain more fully how the cut-off of 19.5% CD44-/CD24- was determined.

9. Figure 1C – please explain how the ratios of different subtypes of breast cancer with high or low CD44-/CD24- were calculated. According to the figure, for luminal cancers, there was a metastasis rate of ~40% in the high group and less then 10% in the low group. How were the numbers of 63.1% and 32.6% derived from Figure 1C?

Then the same figures are provided for Figure 2A, when using the test set. Were the data in Figure 1C from the training set? Why are the final metastasis rates exactly the same? This needs a better explanation.

10. Page 6: reference to Figure S3C should be Figure S2C?

11. Page 7 – Should Figure S5A,B actually be Figure S3?

12. Page 7: The statement: "Moreover, a low CSC percentage led to different risks for developing tumor metastasis 5 years after diagnosis and adjuvant chemotherapy between the C1 and C0 patients (Figure S3D)." This does not appear to apply to Figure S3D and I cannot tell to which figure this statement is referring.

13. Page 7, last sentence: "In the present study, we first designed the experiments illustrated in Figure 3A to characterize the percentages of different cell subtypes among MDA-MB-231 cells using cell culture and flow cytometric cell sorting of parental MDA-MB-468 cells." If I understand correctly, this should re-worded as follows: "In the present study, we first designed the experiments illustrated in Figure 3A and Figure S5 to characterize the percentages of different cell subtypes among MDA-MB-231 and MDA-MB-468 cells using cell culture and flow cytometric cell sorting of parental cells.

14. Figure 3C raises questions about reproducibility of the data. The profile in Figure 3C after 7 days in culture looks very different from that shown in Figure 3B.

15. Figure 5C: please explain how you obtained a weight measurement for the lung metastases. You record lung metastasis weights of up to 2 grams. Lungs typically weight about 0.2 grams. Lungs of 2 grams seem improbable, let alone metastases in lungs weighing 2 grams. Also, tumor sizes of 700 mm2 do not seem possible in lungs.

16. The RNAseq analysis requires more explanation. In Figure 6B, please explain the three columns shown in the heat map. Without their designation, the rest of this figure is hard to interpret. It seems that only one single cell per timepoint has been analysed. Is this correct? How reliable is sequencing from a single cell? Or was the analysis from many cells grown up from a single cell? How many single cells (or derivative of single cells) were analysed at each time point? There is a discrepancy between the protocol described in the text and that provided in the Methods section, when describing the RNAseq analysis. It is difficult to relate the significance of the genes shown in Panel B to the Kaplan-Meier data when it is not clear what the three columns in panel B represent.

17. Figure 6D: I assume that the "*" and "#" are meant to convey some sort of significance, but they do not appear for any of the genes assessed by RT-PCR. Does that mean that none of the changes were significant?

18. Why does the legend for Figure S6 talk about SILAC for protein analysis? Was SILAC run on these samples? Impossible on single cells. No mention of SILAC in Methods.

19. Page 10: Explain why YAP was selected for analysis following knockdown of RHBDL2. Was there a previously known connection between these two genes? For the CD44-/CD24- cells in Figure 7B, was YAP phosphorylation also altered? Please show a western demonstrating that RHBDL2 is reduced after transfection with siRHBDL2.

20. Figure 7D: text says 0.7% CD44+/CD24- but figure says 0.5% and vice versa for the MDA-MB-468 cells.

21. The text on page 11 needs re-writing. The second paragraph, commencing with "We next selected…" is a repeat of the text above where you have already done this step and analysed the cells by flow cytometry.

Reviewer #2:

The manuscript describes a possible link between the presence of CD44-/CD24- cells and post-operative site distant metastasis. This is not a new concept, yet the actual biological function of CSC, particularly of the phenotype CD44-/CD24- remains inconclusive. The primary strength of the study is the large number of patient samples used in the retrospective analysis that provides the rationale for in vitro experiments using cultured cancer cells. Several technical and methodological limitations were identified.

The description of the patient cohort is very elusive. The bioinformatic analysis lacks in rigor as the methodology used to identify and quantitate CD44-/CD24- cells in patients is not properly described. For instance, Figure legends describe the use of flow cytometry for quantification of CD44-/CD24- negative cells from patient samples. Yet only an immunofluorescence staining is described in Results. No Methods description is given to FACS of patient samples. The manuscript seems focused on TNBC, yet the reader has a difficult time understanding how many samples are actually from TNBC patients. Two TNBC established cell lines are mentioned in the manuscript but no rationale is provided as to why some experiments are performed only with MDA-MB-468 and some with MDA-MB-231. The same is true for several other experiments. The extensive molecular description of CD44-/CD24- cell dedifferentiation into CSCs deserves distinction but will need to be confirmed in patient samples before conclusions can be drawn with regards to the biological significance of these findings to breast cancer patients and the medical community at large.

Clarify the methodology used to quantitate CD44-/CD24- in patient samples, as well as the bioinformatic analysis supporting the current hypothesis.

Provide a detailed analysis of clinical and pathologic information of the patient cohort, including histology.

Several references are 10 years old or older, new references supporting the authors finds would strength the hypothesis.

https://doi.org/10.7554/eLife.65418.sa1

Author response

Reviewer #1:

The authors set out to demonstrate the importance of a subset of cancer cells lacking CD44 and CD24, in promoting tumor progression, metastasis and resistance to therapy. They analyse archival breast tissues from patients where the clinical outcome – development of metastasis and therapy response – are known. They then take this information into cell culture experiments, seeking the mechanism that underlies the importance of these CD44-/CD24- cells. Their major premise is that these double negative cells can de-differentiate into cancer stem cells that subsequently promote metastasis and chemotherapy resistance.

The overall concept of this study is good and the cell-based results are mainly clear. However, the archival tissue analysis needs clarification, as does the RNAseq analysis, as detailed below.

We thank the reviewer for his/her positive comments. We also recognize the limitations of our current study and have fully addressed the reviewers’ concerns by clarifying the analysis methods in the revision.

The breast cancer tissues are scored by immunofluorescence for expression of CD44 and CD24, but there is no indication that these two markers are being scored only in tumor cells rather than in all lineages within the tumor microenvironment. This is an important point that must be clarified. Also, the RNAseq analysis is reported to be conducted on single cells, but the data presented do not indicate if this is actually the case and how many replicate samples were analysed and which sample yielded each heat map pattern. Again clarification is required before the data can be interpreted.

We are sorry for the confusion. Actually, we analyzed the frequency of CD44-CD24- breast cancer cells in breast cancer tissue sections, but not stromal, infiltrates and other non-tumor cells, identified in the H&E stained corresponding series of breast cancer sections. Now, we have clarified it in the revision and provided the relevant H&E stained section in the supplementary Figure x. Please see our answers regarding the RNAseq method in question 12.

Finally, the conclusion from this study is that RHBDL2 induces spontaneous conversion of CD44-/CD24- cells into CD44+/CD24- cells via YAP1, USP31 and decreased NFkB signaling. This may well be the case, but has not been proven by the experiments presented here. Whilst DHBDL2 does alter YAP, USP31 and NFkB, it could also be working through an alternate pathway to induce CSCs.

We fully understand his/her concern. It is true that RHBDL2 acts as an intramembrane serine protease that can cleave and activate other substrates, including ephrin B and others, contributing to the development and progression of malignant tumors. Actually, ephrin B has been shown to function as a prognostic factor of uterine cervical (PMID 25205602 ). Unfortunately, we are unable to perform more experiments to test alternative mechanisms underlying the action of RHBDL2 in promoting distant metastasis of breast cancer due to the limited resources. In response to his/her concern, we have modified the conclusion of this study in a conservative manner and briefly discussed the potential alternative mechanisms underlying the action of RHBDL2.

1. Figure 1 and S1 and S2A score the numbers of samples with the varying CD44/CD24 protein levels. For Figure S1, this is shown by immunofluorescence, but there is no indication of whether the cells being analysed are tumor cells or host cells from the microenvironment. There should be co-staining for EpCAM or CK19 to confirm that tumor cells only are being scored. I am assuming that the data shown in Figure S2A are actually from the immunostaining and not from flow cytometry analysis as stated in the figure legend? If this was a flow analysis, there is no description of the origin or processing of the samples reported in Figure 1A for flow cytometry. The legend for Figure S2A states that the data are stratifying only metastatic tumors, but in Figure 1A, apparently many of the tumors showed no metastasis. All these points need to be clarified/corrected.

We fully understand his/her concern. In this study, we performed H&E staining and immunofluorescence simultaneously in a series of consecutive tissue sections. With H&E stained sections, we identified tumor cells and other types of non-tumor cells in the tumor regions. Subsequently, we analyzed the numbers of CD44-CD24- tumor cells in each section as described in the method section to determine the frequency of CD44-CD24- cells in total tumor cells. We have provided the relevant H&E stained sections in the revision and clarified the data in Figure S2A(now is Figure 1—figure supplement 2) by revising the figure legend in the revision. We are sorry for our carelessness to state wrong method in the Figure S2(now is Figure 1—figure supplement 2) legend

2. This first section of the manuscript is poorly presented and I am struggling to follow the arguments and data being provided. Many of the figures are mis-labelled in the text and they are not presented in consecutive order as discussed in the text. For some references to data in the text where the wrong figure has been cited, I cannot even find the correct figure.

We fully understand the reviewer’s concern and have corrected the figure citations accordingly.

3. Possibly the way the data are presented, with some figures referring to the training set and other figures to the test set is adding to the confusion when it is not clearly stated which set is under discussion at the time.

We appreciate his/her advice. Now, we have specified the figures and their descriptions in the revision, accordingly.

4. Late metastasis – better phrased as delayed metastasis.

We appreciate his/her advice. Now, we have changed the “late” into “delayed” metastasis in the revision.

5. The sentence in the Introduction: "Previous studies have shown that CD44+/CD24- breast CSCs might be a dominant factor in relapse of triple negative breast cancer (TNBC), due to their possession of potent self-renewal and differentiation capacities to differentiate into mature CD44-/CD24-, CD44+/CD24+, and CD44-/CD24+ cancer cells (Geng et al., 2014; Wang et al., 2014)". It is not at all clear to me that the references provided support this comment. Geng et al. do not mention CD44-/CD24- cells at all, and Wang et al. do not discuss differentiation of CD44+/CD24- into the other phenotypes. Only CD44- cells are mentioned, not CD24.

We thank the reviewer for his/her careful review. To avoid potential overstatement, we have modified the sentence into a general concept by deleting specific differential phenotypes in the revision.

6. Sentence in Introduction: "Indeed, injection of up to 1000 breast CSCs was able to generate a solid tumor mass in immunocompromised mice (Chaffer et al., 2011; Iliopoulos et al., 2011)." Please clarify. It is not obvious to me that Chaffer et al. specified how many CSCs they injected and Iliopoulos et al. showed that as few as 50 CSCs could form tumors.

We thank the reviewer for his/her careful review. We have re-checked the references and found that Chaffer et al. described that injection of up to 50 breast CSCs was able to generate a solid tumor mass in immunocompromised mice. Accordingly, we would like to keep the sentence as is.

7. Sentence in Introduction: "Moreover, previous studies reported that non-CSCs, such as CD44-/CD24- TNBC cells, are able to spontaneously convert into CSCs to renew the CSC pool, resulting in chemoresistance (Gruber et al., 2016; Kim et al., 2015; Ye et al., 2018)." The statement being referenced is for non-CSCs in TNBC, but Gruber et al. and Ye et al. are not reporting on TNBC.

We thank the reviewer for his/her careful review. Now, we have revised the text by changing “CD44-/CD24- TNBC cells” to “CD44-/CD24- breast cancer cells” to reflect the findings in the references.

8. Figure 1B – please explain more fully how the cut-off of 19.5% CD44-/CD24- was determined.

We thank the reviewer for his/her advice. the average frequency of CD44-/CD24- cancer cells in all samples was 19.7% and the median was 19.5%. The receiver-operating characteristic curve analysis also showed a decision threshold of 19.5% CD44-/CD24- cancer cells; thus, we used this cut-off point to perform a subgroup analysis.

9. Figure 1C – please explain how the ratios of different subtypes of breast cancer with high or low CD44-/CD24- were calculated. According to the figure, for luminal cancers, there was a metastasis rate of ~40% in the high group and less than 10% in the low group. How were the numbers of 63.1% and 32.6% derived from Figure 1C?

Figure 1C describes the effect of CD44-/CD24- levels on the metastasis rates among three molecular subtypes of breast cancer, i.e., 63.79% vs. 22.22% (high vs. low CD44-/CD24- tumor cells in TNBC), 40.85% vs. 6.82% (luminal), and 47.06% vs. 13.64% (HER-2).

Then the same figures are provided for Figure 2A, when using the test set. Were the data in Figure 1C from the training set? Why are the final metastasis rates exactly the same? This needs a better explanation.

Figure 2A contains an error in the description and has been corrected to luminal: 22.73% vs. 8.89%; HER-2: 50% vs. 18.42%; TNBC: 41.67% vs. 15% for high vs. low CD44-/CD24- cells.

10. Page 6: reference to Figure S3C should be Figure S2C?

We thank the reviewer for their careful review and have corrected this figure citation.

11. Page 7 – Should Figure S5A,B actually be Figure S3?

We thank the reviewer for their careful review and have corrected the figure citation of Figure S5A,B to Figure S4A,B(now is Figure 2—figure supplement 1).

12. Page 7: The statement: "Moreover, a low CSC percentage led to different risks for developing tumor metastasis 5 years after diagnosis and adjuvant chemotherapy between the C1 and C0 patients (Figure S3D)." This does not appear to apply to Figure S3D and I cannot tell to which figure this statement is referring.

We are sorry for the confusion. The correct description should be that we stratified patients with <2% CD44+/CD24- and >19.5% CD44-/CD24- tumor cells in the C1 group while those with <2% CD44+/CD24- and <19.5% CD44-/CD24- tumor cells in the C0 group. We have clarified it in the text and figure legend accordingly.

13. Page 7, last sentence: "In the present study, we first designed the experiments illustrated in Figure 3A to characterize the percentages of different cell subtypes among MDA-MB-231 cells using cell culture and flow cytometric cell sorting of parental MDA-MB-468 cells." If I understand correctly, this should re-worded as follows: "In the present study, we first designed the experiments illustrated in Figure 3A and Figure S5 to characterize the percentages of different cell subtypes among MDA-MB-231 and MDA-MB-468 cells using cell culture and flow cytometric cell sorting of parental cells.

We thank the reviewer for his/her advice. Now, we have revised it in the manuscript accordingly. The data in Figure 3A, B have been updated with data of primary cells from breast cancer patients.

14. Figure 3C raises questions about reproducibility of the data. The profile in Figure 3C after 7 days in culture looks very different from that shown in Figure 3B.

We are sorry for the confusion. Actually, the Figure 3C shows the flow cytometric data of primary tumor cell-derived tumor in nude mice, which is different from the flow cytometric chart of the cultured cell lines. We have specified it in the Figure 3C legend.

15. Figure 5C: please explain how you obtained a weight measurement for the lung metastases. You record lung metastasis weights of up to 2 grams. Lungs typically weight about 0.2 grams. Lungs of 2 grams seem improbable, let alone metastases in lungs weighing 2 grams. Also, tumor sizes of 700 mm2 do not seem possible in lungs.

We are sorry for our carelessness. Actually, we measured the lung weights, but not the tumor weights because we cannot dissect all metastatic tumor nodules in the lung. Similarly, the tumor size data were calculated wrongly, based on the magnified images. Now, we have corrected these mistakes in the revision. Overall, our data indicated the converted CSCs had similar tumorigenicity and differential capacity in mice.

16. The RNAseq analysis requires more explanation. In Figure 6B, please explain the three columns shown in the heat map. Without their designation, the rest of this figure is hard to interpret. It seems that only one single cell per timepoint has been analysed. Is this correct? How reliable is sequencing from a single cell? Or was the analysis from many cells grown up from a single cell? How many single cells (or derivative of single cells) were analysed at each time point? There is a discrepancy between the protocol described in the text and that provided in the Methods section, when describing the RNAseq analysis. It is difficult to relate the significance of the genes shown in Panel B to the Kaplan-Meier data when it is not clear what the three columns in panel B represent.

We are sorry that we did not describe the experimental protocol in detail and present the data in Figure 6B well. We cultured CD44-/CD24- MDA-MB-231 cells in a single cell manner for 24, 72 and 120 h and they were harvested for RNAseq analysis of three single cells at each time point. We have modified the experimental protocol to specify it in the revision. Although we did single cell RNAseq the data could not be explained by single cell RNAseq, rather than general RNAseq. Accordingly, we changed the term of single cell RNAseq into RNAseq to avoid potential misleading in the revision. From the data in the heatmap, the repeated single samples display similar levels of each gene, indicative of its reliable nature. We are sorry for our carelessness and now, we have modified the manuscript to ensure the consistence between the protocol and result sections. In addition, we have labeled the columns in Figure 6B to specific the samples and time points.

17. Figure 6D: I assume that the "*" and "#" are meant to convey some sort of significance, but they do not appear for any of the genes assessed by RT-PCR. Does that mean that none of the changes were significant?

We are sorry for the confusion. To clarify the significance among groups of cells, we have specified the significance as * or # vs. CD44-/CD24- cells in the Figure legend. There were significant difference in the levels of mRNA transcripts of some genes between these types of cells in our experimental system.

18. Why does the legend for Figure S6 talk about SILAC for protein analysis? Was SILAC run on these samples? Impossible on single cells. No mention of SILAC in Methods.

We are sorry for our mistake. We did not run SILAC in this study. We have deleted it in the revision. (now is Figure 6—figure supplement 1)

19. Page 10: Explain why YAP was selected for analysis following knockdown of RHBDL2. Was there a previously known connection between these two genes? For the CD44-/CD24- cells in Figure 7B, was YAP phosphorylation also altered? Please show a western demonstrating that RHBDL2 is reduced after transfection with siRHBDL2.

We thank the reviewer for his/her valuable comments. Indeed, the Hippo pathway is important for the stemness of tumor cells. Thus, we selected the YAP1 as a verification indicator. We did not find any available data on how RHBDL2 modulates YAP1 expression and phosphorylation in any type of tumors in the literature. In response to his/her concerns, we have performed additional experiments and found that transfection with RHDBL2-specific siRNAA effectively decreased the relative levels of its expression in CD44-/CD24- cells. More importantly, RHDBL2 silencing also decreased YAP1 expression, but increased YAP1 phosphorylation (Figure 7B), leading to increased USP31 expression and decreased NF-κB phosphorylation in CD44-/CD24- breast cancer cells. These data supported our conclusion.

20. Figure 7D: text says 0.7% CD44+/CD24- but figure says 0.5% and vice versa for the MDA-MB-468 cells.

We are sorry for the confusion. Actually, the Figure 7D describes the data from MDA-MB-231 cells, which were 0.5% of CD44+/CD24- cells while the Figure 7E displays the data (0.7% of CD44+/CD24- cells) from MDA-MB-468 cells following RHDBL2 silencing. Now,we have clarified the data in the revision.

21. The text on page 11 needs re-writing. The second paragraph, commencing with "We next selected…" is a repeat of the text above where you have already done this step and analysed the cells by flow cytometry.

We thank the reviewer for their careful review and have revised the paragraph accordingly.

Reviewer #2:

The manuscript describes a possible link between the presence of CD44-/CD24- cells and post-operative site distant metastasis. This is not a new concept, yet the actual biological function of CSC, particularly of the phenotype CD44-/CD24- remains inconclusive. The primary strength of the study is the large number of patient samples used in the retrospective analysis that provides the rationale for in vitro experiments using cultured cancer cells. Several technical and methodological limitations were identified.

The description of the patient cohort is very elusive. The bioinformatic analysis lacks in rigor as the methodology used to identify and quantitate CD44-/CD24- cells in patients is not properly described. For instance, Figure legends describe the use of flow cytometry for quantification of CD44-/CD24- negative cells from patient samples. Yet only an immunofluorescence staining is described in Results. No Methods description is given to FACS of patient samples. The manuscript seems focused on TNBC, yet the reader has a difficult time understanding how many samples are actually from TNBC patients. Two TNBC established cell lines are mentioned in the manuscript but no rationale is provided as to why some experiments are performed only with MDA-MB-468 and some with MDA-MB-231. The same is true for several other experiments. The extensive molecular description of CD44-/CD24- cell dedifferentiation into CSCs deserves distinction but will need to be confirmed in patient samples before conclusions can be drawn with regards to the biological significance of these findings to breast cancer patients and the medical community at large.

We fully understand the reviewer’s concerns and we are sorry for the confusion in the manuscript. As we addressed the similar concerns from the reviewer 1, we have clarified the experimental protocols and corrected couple mistakes in the revision. Actually, we used H&E staining to identify tumor cells in breast cancer tissue sections and employed immunofluorescence to quantify the number of CD44-/CD24- tumor cells in the consecutive sections to determine the frequency of CD44-/CD24- breast cancer cells in total tumor cells in individual tissue sections. Moreover, Supplementary file 1A (Table S1) shows the number of patients with three molecular classifications and their clinical information. In addition, our data have been verified with two cell lines MDA-MB-468 and MDA-MB-231, and these data have been added to the manuscript. In order to confirm our findings in patients’ samples, we utilized fresh specimens from breast cancer patients to isolate primary cells for data verification. The corresponding results have been added in Figure 3.

Clarify the methodology used to quantitate CD44-/CD24- in patient samples, as well as the bioinformatic analysis supporting the current hypothesis.

We appreciate his/her advice. As we stated above, we have clarified the methods for quantifying CD44-/CD24- breast cancer cells in tissue sections above and in the revision. In addition, we have modified the section of bioinformatics to clarify the strategies for anlaysis of data in the revision.

Provide a detailed analysis of clinical and pathologic information of the patient cohort, including histology.

We appreciate his/her advice. Now, we have provided the detailed information on demographic and clinical characteristics of all patients we studied in Supplementary file 1A (Table S1) of the revision.

Several references are 10 years old or older, new references supporting the authors finds would strength the hypothesis.

We appreciate his/her advice. Now, have updated the reference section with new references available in the literature.

https://doi.org/10.7554/eLife.65418.sa2

Article and author information

Author details

  1. Xinbo Qiao

    Department of Oncology, Shengjing Hospital, China Medical University, Shenyang, China
    Contribution
    Data curation, Formal analysis, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Yixiao Zhang, Lisha Sun and Qingtian Ma
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6759-921X
  2. Yixiao Zhang

    1. Department of Oncology, Shengjing Hospital, China Medical University, Shenyang, China
    2. Dapartment of Urology, Shengjing Hospital, China Medical University, Shenyang, China
    Contribution
    Validation, Methodology
    Contributed equally with
    Xinbo Qiao, Lisha Sun and Qingtian Ma
    Competing interests
    No competing interests declared
  3. Lisha Sun

    Department of Oncology, Shengjing Hospital, China Medical University, Shenyang, China
    Contribution
    Resources, Software, Writing - review and editing
    Contributed equally with
    Xinbo Qiao, Yixiao Zhang and Qingtian Ma
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4095-5026
  4. Qingtian Ma

    Department of Oncology, Shengjing Hospital, China Medical University, Shenyang, China
    Contribution
    Formal analysis, Writing - original draft, Project administration
    Contributed equally with
    Xinbo Qiao, Yixiao Zhang and Lisha Sun
    Competing interests
    No competing interests declared
  5. Jie Yang

    Department of Oncology, Shengjing Hospital, China Medical University, Shenyang, China
    Contribution
    Software, Investigation
    Competing interests
    No competing interests declared
  6. Liping Ai

    Department of Oncology, Shengjing Hospital, China Medical University, Shenyang, China
    Contribution
    Data curation, Formal analysis
    Competing interests
    No competing interests declared
  7. Jinqi Xue

    Department of Oncology, Shengjing Hospital, China Medical University, Shenyang, China
    Contribution
    Formal analysis
    Competing interests
    No competing interests declared
  8. Guanglei Chen

    Department of Oncology, Shengjing Hospital, China Medical University, Shenyang, China
    Contribution
    Formal analysis
    Competing interests
    No competing interests declared
  9. Hao Zhang

    1. Department of Oncology, Shengjing Hospital, China Medical University, Shenyang, China
    2. Department of Breast Surgery, Liaoning Cancer Hospital and Institute, Cancer Hospital of China Medical University, Shenyang, China
    Contribution
    Data curation, Software
    Competing interests
    No competing interests declared
  10. Ce Ji

    1. Department of Oncology, Shengjing Hospital, China Medical University, Shenyang, China
    2. Department of General Surgery, Shengjing Hospital, China Medical University, Shenyang, China
    Contribution
    Formal analysis, Funding acquisition
    Competing interests
    No competing interests declared
  11. Xi Gu

    Department of Oncology, Shengjing Hospital, China Medical University, Shenyang, China
    Contribution
    Formal analysis
    Competing interests
    No competing interests declared
  12. Haixin Lei

    Institute of Cancer Stem Cell, Cancer Center, Dalian Medical University, Dalian, China
    Contribution
    Writing - review and editing
    Competing interests
    No competing interests declared
  13. Yongliang Yang

    Center for Molecular Medicine, School of Life Science and Biotechnology, Dalian University of Technology, Dalian, China
    Contribution
    Writing - review and editing
    Competing interests
    No competing interests declared
  14. Caigang Liu

    Department of Oncology, Shengjing Hospital, China Medical University, Shenyang, China
    Contribution
    Conceptualization, Writing - review and editing
    For correspondence
    angel-s205@163.com
    Competing interests
    Reviewing Editor, eLife
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2083-235X

Funding

National Science Foundation (#81572609)

  • Caigang Liu

Funders have no role in research design, data collection and decisions to interpret or submit works for publication.

Acknowledgements

This work was supported by grants from The National Natural Science Foundation of China (#81572609), the Major Project Construction Foundation of China Medical University (#2017ZDZX05) and Liaoning Province young top talent project (#XLYC1807099).

Ethics

Animal experimentation: The experimental protocol was approved by the Animal Research and Care Committee of China Medical University (Shenyang, China) and followed the Guidelines of the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research. Female BALB/c nude mice (6 weeks old) were obtained from Human Silaikejingda Laboratory Animals (Changsha, China) and housed in a specific pathogen-free facility with free access to autoclaved food and water. All surgery was performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering. The current study was approved by the Ethics Committee of all three hospital review board review boards ((Project identification code: Project identification code: 2018PS304K, date on 03/05/2018 2018PS304K, date on 03/05/2018)).

Senior Editor

  1. Mone Zaidi, Icahn School of Medicine at Mount Sinai, United States

Reviewing Editor

  1. Renata Pasqualini, Rutgers University, United States

Reviewer

  1. Robin Anderson, Olivia Newton-John Cancer Research Institute, Australia

Publication history

  1. Received: December 3, 2020
  2. Accepted: July 25, 2021
  3. Accepted Manuscript published: July 28, 2021 (version 1)
  4. Version of Record published: August 6, 2021 (version 2)

Copyright

© 2021, Qiao et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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