Anti-tumor drug resistance is a challenge for human triple-negative breast cancer (TNBC) treatment. Our previous work demonstrated that TNFAIP2 activates RAC1 to promote TNBC cell proliferation and migration. However, the mechanism by which TNFAIP2 activates RAC1 is unknown. In this study, we found that TNFAIP2 interacts with IQGAP1 and Integrin β4. Integrin β4 activates RAC1 through TNFAIP2 and IQGAP1 and confers DNA damage-related drug resistance in TNBC. These results indicate that the Integrin β4/TNFAIP2/IQGAP1/RAC1 axis provides potential therapeutic targets to overcome DNA damage-related drug resistance in TNBC.
This study presents a rather valuable finding that IQGAP1 interacts with TNFAIP2, which activates Rac1 to promote drug resistance in TNBC. The evidence supporting the claims of the authors is quite solid. The work will be of interest to scientists working on breast cancer.https://doi.org/10.7554/eLife.88483.3.sa0
Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in women (Bray et al., 2018). Although the diagnosis and treatment of breast cancer has entered the era of molecular typing, 35% of breast cancers still experience recurrence, metastasis, and treatment failure (Zhao et al., 2020). According to the expression of estrogen receptor (ERα), progesterone receptor (PR), and human epidermal growth factor receptor (HER2), breast cancer is divided into ER/PR-positive, HER2-positive, and triple-negative breast cancer (TNBC) (Sotiriou and Pusztai, 2009). For ER/PR- and HER2-positive breast cancer, endocrine therapies such as tamoxifen and anti-HER2 targeted therapy such as trastuzumab have achieved good efficacy. Targeted drugs for TNBC patients with BRCA1/2 mutations include two poly ADP-ribose polymerase (PARP) inhibitors, olaparib and talazoparib. These targeted drugs cannot fully meet the clinical needs of patients with various TNBC subtypes (Bai et al., 2021). Currently, DNA damage chemotherapy drugs, including epirubicin and cisplatin, are widely used for TNBC treatment.
TNBC often recurs and metastasizes due to the development of chemoresistance, although it is initially responsive to chemotherapeutic drugs (Jamdade et al., 2015). Chemoresistance severely impacts the clinical outcomes of patients. Tumor cells become resistant to chemotherapeutic agents through several mechanisms, such as improving DNA damage repair, changing the intracellular accumulation of drugs, or increasing anti-apoptotic mechanisms (Hill et al., 2019). Therefore, characterization of the underlying molecular mechanisms by which resistance occurs will provide opportunities to develop precise therapies to enhance the efficacy of standard chemotherapy regimens (Longley and Johnston, 2005; Rincón et al., 2016).
TNFAIP2 is abnormally highly expressed in a variety of tumor cells, including TNBC (Jia et al., 2016), nasopharyngeal carcinoma (Chen et al., 2011), malignant glioma (Cheng et al., 2015), uroepithelial carcinoma (Niwa et al., 2019), and esophageal squamous cell carcinoma (Xie and Wang, 2017), and is associated with poor prognosis. Our previous work (Jia et al., 2016; Jia et al., 2018) showed that TNFAIP2, as a KLF5 downstream target protein, can interact with RAC1 (Didsbury et al., 1989), a member of the Rho small GTP enzyme family, and activate RAC1 to alter the cytoskeleton, thereby inducing filopodia and lamellipodia formation and promoting the adhesion, migration, and invasion of TNBC cells. After activation, RAC1 can activate AKT, PAKs, NADPH oxidase, and other related signaling pathways to promote cell survival, proliferation, adhesion, migration, and invasion (Rul et al., 2002). Activation of RAC1 can reduce the therapeutic response to trastuzumab in breast cancer and increase the resistance of TNBC cells to paclitaxel (Liu et al., 2019), but the specific mechanism of action is not completely clear.
RAC1 has been shown to play an important role in DNA damage repair. Activated RAC1 can promote the phosphorylation of the DNA damage response-related molecules ATM/ATR, CHK1/2, and H2AX by activating the activity of protein kinases such as ERK1/2, JNK, and p38 (Yan et al., 2012; Wu et al., 2019), thus improving the DNA damage repair ability and inhibiting tumor cell apoptosis (Hu et al., 2016; Li et al., 2020; Hervieu et al., 2020). RAC1 also promotes aldolase release and activation by changing the cytoskeleton and activates the ERK pathway to increase the pentose phosphate pathway to promote nucleic acid synthesis, providing more raw materials for DNA damage repair (Li et al., 2021; Feng et al., 2010). At the same time, the interaction of RAC1 and PI3K promoted AKT phosphorylation and glucose uptake (Higuchi et al., 2008; Hu et al., 2018). Therefore, RAC1 is well established to promote the chemoresistance of breast cancer by promoting DNA damage repair.
Integrin β4 (ITGB4) is a major component of hemidesmosomes and a receptor molecule of laminin. Studies have shown that laminin-5 interacts with ITGB4 to activate RAC1 activity and promote cell migration (Hamill et al., 2009) and polarization (Wu et al., 2018) by altering the cytoskeleton. Since ITGB4-positive cancer stem cell (CSC)-enriched mesenchymal cells were found to reside in an intermediate epithelial/mesenchymal phenotypic state, ITGB4 can be used to enable stratification of mesenchymal-like TNBC cells (Bierie et al., 2017). In addition, the expression of ITGB4 on ALDHhigh breast cancer and head and neck cancer cells was significantly greater than that on ALDHlow cells, proving the effects that ITGB4 targets on both bulk and CSC populations (Ruan et al., 2020). Furthermore, ITGB4-overexpressing TNBC cells provided cancer-associated fibroblasts (CAFs) with ITGB4 proteins via exosomes, and ITGB4-overexpressing CAF-conditioned medium promoted the proliferation, epithelial-to-mesenchymal transition, and invasion of breast cancer cells (Sung et al., 2020). ITGB4 also promotes breast cancer cell resistance to tamoxifen-induced apoptosis by activating the PI3K/AKT signaling pathway and promotes breast cancer cell resistance to anoikis by activating RAC1 (Kim et al., 2012). However, how ITGB4 activates RAC1 is not completely clear.
RAC1 activity is regulated by guanylate exchange factors (GEFs), GTPase activation proteins (GAPs), and guanine separation inhibitors (GDIs) (Cherfils and Zeghouf, 2013). GAPs typically provide the necessary catalytic groups for GTP hydrolysis, but not all GAPs function as hydrolases. IQGAP1 lacks an arginine in the GTPase binding domain and cannot exert the hydrolysis effect of GAPs (Schmidt, 2012). IQGAP1 can increase the activity of RAC1 and CDC42 (Smith et al., 2015; Gorisse et al., 2020).
In this study, we demonstrated that TNFAIP2 interacts with IQGAP1 and ITGB4. ITGB4 promotes TNBC drug resistance via the TNFAIP2/IQGAP1/RAC1 axis by promoting DNA damage repair. Our results suggest that ITGB4 and TNFAIP2 might serve as promising therapeutic targets for TNBC.
To explore the functional significance of TNFAIP2 in TNBC drug resistance, we constructed stable TNFAIP2 overexpression and TNFAIP2 knockdown HCC1806 and HCC1937 cells. As shown in Figure 1A–E, overexpression of TNFAIP2 significantly increased cell viability when treated with Epirubicin (EPI) and Talazoparib (BMN). Additionally, knockdown of TNFAIP2 significantly decreased cell viability when treated with EPI and BMN (Figure 1F–J). We then examined the effects of TNFAIP2 on DNA damage repair and found that TNFAIP2 promotes DNA damage repair in response to EPI and BMN. TNFAIP2 overexpression decreased the protein expression levels of γH2AX, a marker of DNA damage, and cleaved-PARP, a marker of apoptosis (Figure 1K). Additionally, knockdown of TNFAIP2 significantly increased γH2AX and cleaved-PARP protein expression levels in response to EPI and BMN in both cell lines (Figure 1L).
The function of TNFAIP2 was further validated by using two other DNA damage drugs, DDP and AZD (Figure 1—figure supplement 1A–L). These results suggested that TNFAIP2 enhances TNBC cell drug resistance by promoting DNA damage repair.
To test whether TNFAIP2 also decreases the sensitivity of TNBC cells to EPI and BMN in vivo, we performed animal experiments in nude mice. HCC1806 cells with stable TNFAIP2 knockdown were orthotopically inoculated into the fat pad of 7-week-old female mice (n = 8 or 12/group). Western blotting (WB) was performed to detect the knockdown effect of TNFAIP2 protein in animal experiments (Figure 2—figure supplement 2G). When the tumor mass reached approximately 50 mm3, each group was divided into two subgroups to receive either EPI (2.5 mg/kg, twice a week) or vehicle control for 23 days and either BMN (1 mg/kg, twice a week) or vehicle control for 29 days. We observed that depletion of TNFAIP2 suppressed breast cancer cell growth in vivo. This is consistent with our previous report (Jia et al., 2016). More importantly, TNFAIP2 depletion further decreased tumor volume when mice were treated with EPI and BMN (Figure 2A–F). Meanwhile, BMN treatment had no effect on the body weight of mice (Figure 2—figure supplement 1F). Consistently, EPI and DDP generated similar results but decreased mouse body weight due to their high toxicity (Figure 2—figure supplement 1D, E). These results suggest that inhibition of TNFAIP2 expression can overcome HCC1806 breast cancer cell drug resistance in animals.
Since chemotherapeutic agents and PRAP inhibitors induce DNA damage directly or indirectly, DNA damage repair ability profoundly affects the sensitivity of cancer cells to these therapies (Woods and Turchi, 2013; Cheung-Ong et al., 2013). Since TNFAIP2 can activate RAC1, a well-known drug resistance protein, we investigated whether TNFAIP2 induces chemotherapeutic resistance through RAC1. We found that RAC1 knockdown abrogated the effects of TNFAIP2 overexpression-induced drug resistance to EPI and BMN in HCC1806 and HCC1937 cells (Figure 3A–F). We also found that γH2AX and cleaved-PARP protein levels were upregulated again in RAC1 knockdown and TNFAIP2-overexpressing HCC1806 and HCC1937 cells in response to EPI and BMN (Figure 3G–J). We obtained similar results by using DDP and AZD treatment (Figure 3—figure supplement 1A–J). Collectively, these results suggest that TNFAIP2 promotes DNA damage repair and drug resistance via RAC1.
To characterize the mechanism by which TNFAIP2 activates RAC1, we performed an immunoprecipitation and silver staining (IP-MS) experiment. We found that TNFAIP2 interacts with IQGAP1 (Figure 4A). To validate whether TNFAIP2 interacts with IQGAP1, we constructed HCC1806 cells with stable Flag-TNFAIP2 overexpression and collected Flag-tagged TNFAIP2 cell lysates for immunoprecipitation assays using Flag-M2 beads (Figure 4—figure supplement 1A).We performed immunoprecipitation using ananti-IQGAP1 antibody and found that endogenous IQGAP1 protein interacted with endogenous TNFAIP2 protein inHCC1806 cells (Figure 4B). Next, we mapped the regions of TNFAIP2 and IQGAP1 proteins responsible for the interaction by generating a series of Flag-TNFAIP2 deletion mutants and transfected them into HEK293T cells together with full-length IQGAP1. Then, we performed immunoprecipitation assays using Flag-M2 beads (Figure 4—figure supplement 1B). We demonstrated that the N-terminus (1–79 aa) of the TNFAIP2 protein interacted with IQGAP1. To explore the function of IQGAP1 in TNBC drug resistance, we knocked down IQGAP1 in HCC1806 and HCC1937 cells. As shown in Figure 4C–G, knockdown of IQGAP1 significantly decreased cell viability in the presence of EPI and BMN in both cell lines. We also examined the effects of IQGAP1 on DNA damage repair and found that IQGAP1 promotes DNA damage repair. IQGAP1 knockdown increased γH2AX and cleaved-PARP protein expression levels when HCC1806 and HCC1937 cells were treated with EPI and BMN (Figure 4H). Next, we found that IQGAP1 knockdown abrogated the effects of TNFAIP2 overexpression on resistance to EPI and BMN (Figure 4I–K, Figure 4—figure supplement 1C–E). We also found that γH2AX and cleaved-PARP protein levels were upregulated in IQGAP1 knockdown and TNFAIP2-overexpressing HCC1806 and HCC1937 cells (Figure 4L). In addition, we found that the TNFAIP2 overexpression-induced increase in RAC1 activity was abolished by IQGAP1 knockdown (Figure 4M).
In addition to IQGAP1, TNFAIP2 may interact with ITGB4 (Figure 4A). To validate whether TNFAIP2 interacts with ITGB4, we immunoprecipitated exogenous Flag-tagged TNFAIP2 proteins from HCC1806 cells by using Flag-M2 beads and detected endogenous ITGB4 proteins (Figure 5A).To further confirm the protein‒protein interaction between endogenous TNFAIP2 and ITGB4 proteins, we collected HCC1806 cell lysates and performed immunoprecipitation using an anti-TNFAIP2/ITGB4 antibody and found that endogenous TNFAIP2/ITGB4 protein interacted with endogenous ITGB4/TNFAIP2 protein (Figure 5B, C). We further mapped the regions of TNFAIP2 and ITGB4 proteins responsible for the interaction (Figure 5—figure supplement 2I) by generating a series of Flag-TNFAIP2/GST-fused TNFAIP2 deletion mutants and transfected them into HEK293T cells together with full-length GST-fused ITGB4/ITGB4. Then, we performed immunoprecipitation assays using Flag-M2 beads and glutathione beads. As shown in Figure 5—figure supplement 2J, K, the N-terminus (218–287 aa) of the TNFAIP2 (TNFAIP2-S-N1-3) protein interacted with ITGB4. To map the domains of ITGB4 that interact with TNFAIP2, we transfected Flag-tagged full-length TNFAIP2 into HEK293T cells with full-length or truncated ITGB4. We found that the C-terminus (710–740 aa) of the ITGB4 protein interacted with TNFAIP2 (Figure 5—figure supplement 2L, M). Taken together, these results suggest that TNFAIP2 interacts with ITGB4 and that their interaction is mediated through the N-terminus of TNFAIP2 and the C-terminus of ITGB4.
To explore the function of ITGB4 in TNBC drug resistance, we knocked down ITGB4 in HCC1806 and HCC1937 cells. As shown in Figure 5D–I, knockdown of ITGB4 significantly decreased cell viability in the presence of EPI and BMN in both cell lines. Knockdown of ITGB4 also suppressed HCC1806 xenograft growth in vivo. The knockdown effect of ITGB4 protein in animal experiments was confirmed by WB (Figure 5—figure supplement 2N). More importantly, ITGB4 knockdown further decreased tumor volume when mice were treated with EPI and BMN (Figure 5J–N). Meanwhile, BMN treatment had no effect on the body weight of mice, but EPI treatment decreased mouse body weight due to its toxicity (Figure 5—figure supplement 1H). We then examined the effects of ITGB4 on DNA damage repair and found that ITGB4 promotes DNA damage repair in response to EPI and BMN. ITGB4 knockdown increased γH2AX and cleaved-PARP protein expression levels when HCC1806 and HCC1937 cells were treated with EPI and BMN (Figure 5O). Furthermore, the function of ITGB4 was validated by using two other drugs, DDP and AZD (Figure 5—figure supplement 1A–G). These results suggested that ITGB4 increases TNBC drug resistance and promotes DNA damage repair.
It is well known that ITGB4 can activate RAC1 (Hamill et al., 2009) and that TNFAIP2 interacts with RAC1 and activates it (Jia et al., 2016). To test whether ITGB4 activates RAC1 through TNFAIP2, we measured the levels of GTP-bound RAC1 in ITGB4-overexpressing and ITGB4-knockdown cells. Overexpression of ITGB4 significantly increased the levels of GTP-bound RAC1 in both HCC1806 and HCC1937 cells (Figure 6A). In agreement with this observation, knockdown of ITGB4 significantly decreased the levels of GTP-bound RAC1 in both cell lines (Figure 6B). Next, we knocked down TNFAIP2 in ITGB4-overexpressing HCC1806 and HCC1937 cells and found that ITGB4-increased RAC1 activity was blocked by TNFAIP2 knockdown (Figure 6C, D). Collectively, these results demonstrate that ITGB4 activates RAC1 through TNFAIP2.
It has been reported that RAC1 activity is promoted by IQGAP1 (Schmidt, 2012) and that TNFAIP2 activates RAC1 through IQGAP1 (Figure 4P). We wondered whether ITGB4 activates RAC1 through IQGAP1; therefore, we knocked down IQGAP1 in HCC1806 and HCC1937 cells with stable overexpression of ITGB4 and found that the ITGB4-induced increase in RAC1 activity was abolished by IQGAP1 knockdown (Figure 6E, F). These results suggest that ITGB4 activates RAC1 through TNFAIP2 and IQGAP1.
Since ITGB4, TNFAIP2, and IQGAP1 promote drug resistance by promoting DNA damage repair in TNBC, we wondered whether ITGB4 promoted drug resistance through the TNFAIP2/IQGAP1/RAC1 axis. We knocked down TNFAIP2, IQGAP1, and RAC1 in ITGB4-overexpressing cells and found that blocking the TNFAIP2/IQGAP1/RAC1 axis increased the sensitivity of ITGB4-overexpressing HCC1806 (Figure 7A–I) and HCC1937 cells to EPI and BMN (Figure 7—figure supplement 2O–W). We also found that γH2AX and cleaved-PARP levels were upregulated in TNFAIP2/IQGAP1/RAC1 knockdown HCC1806 and HCC1937 cells stably expressing ITGB4 in the presence of EPI and BMN (Figure 7J–L, Figure 7—figure supplement 2X–Z). DDP and AZD treatment generated similar results (Figure 7—figure supplement 1A–N). Together, these results suggest that ITGB4 promotes DNA damage repair and drug resistance via the TNFAIP2/IQGAP1/RAC1 axis.
To test whether ITGB4 and TNFAIP2 are co-expressed in TNBC, we collected 135 TNBC specimens for immunohistochemistry (IHC) (the IQGAP1 antibody did not work for IHC). Specimens were obtained from the Department of Pathology, Henan Provincial People’s Hospital, Zhengzhou University, China. We performed IHC analyses on two breast cancer tissue chips containing a total of 135 patients with TNBC (Figure 8A–D). TNFAIP2 and ITGB4 protein expression levels were significantly positively correlated (Figure 8E).
Chemotherapies, including EPI and DDP, are the main choice for TNBC patients. Unfortunately, TNBC frequently develops resistance to chemotherapy (Kim et al., 2018). Currently, PARP inhibitors are effective for TNBC with BRCA1/2 mutation or homologous recombination deficiency (HRD) (Noordermeer and van Attikum, 2019; Geenen et al., 2018; Lee and Djamgoz, 2018). PARP inhibitors can cause DNA damage repair defects and have synergistic lethal effects with HRD. Meanwhile, chemotherapy and PARP inhibitor resistance is also a major problem in the clinic.
In this study, we first found that TNFAIP2 promotes TNBC drug resistance and DNA damage repair through RAC1. Next, we found that TNFAIP2 interacts with IQGAP1and ITGB4. We verified that ITGB4 promotes TNBC drug resistance and DNA damage repair through the TNFAIP2/IQGAP1/RAC1 axis. Interestingly, we discovered for the first time that ITGB4 and TNFAIP2 promote RAC1 activity through IQGAP1. Our study reveals that ITGB4 promotes TNBC resistance through TNFAIP2-, IQGAP1-, and RAC1-mediated DNA damage repair (Figure 7). This study provides new targets for reversing TNBC resistance.
ITGB4 is well known to promote breast cancer stemness and can be activated by laminin-5 (Campbell et al., 2018). In addition, ITGB4 is generally in partner with ITGA6, which is another marker of breast CSCs (Ali et al., 2011) and drug resistance (Campbell et al., 2018). Therefore, whether ITGA6 has similar functions needs further study. It was reported that ITGB4 activates RAC1 (Friedland et al., 2007), but the mechanism is unclear. For the first time, we revealed that ITGB4 activates RAC1 through TNFAIP2 and IQGAP1. More importantly, ITGB4 promotes drug resistance through the TNFAIP2/IQGAP1/RAC1 axis.
TNFAIP2 plays important roles in different cellular and physiological processes, including cell proliferation, adhesion, migration, membrane TNT formation, angiogenesis, inflammation, and tumorigenesis (Jia et al., 2018). We previously found that TNFAIP2 was regulated by KLF5 and interacted with the small GTPases RAC1 and CDC42, thereby regulating the actin cytoskeleton and cell morphology in breast cancer cells (Jia et al., 2016). However, the detailed mechanism is not clearly understood. In this study, we found that IQGAP1 mediates this process. IQGAP1 is a crucial regulator of cancer development by scaffolding and facilitating different oncogenic pathways, especially RAC1/CDC42, thus affecting proliferation, adhesion, migration, invasion, and metastasis (Wei and Lambert, 2021). In addition, IQGAP1 is increased during the differentiation of ovarian CSCs and promotes aggressive behaviors (Huang et al., 2015). In our study, we found that TNFAIP2 interacts with IQGAP1 and thus activates RAC1 to induce chemotherapy and PARP inhibitor drug resistance.
Furthermore, TNFAIP2 was reported to induce epithelial-to-mesenchymal transition and confer platinum resistance in urothelial cancer cells (Niwa et al., 2019), and in embryonic stem cell (ESC) differentiation, TNFAIP2 was found to be important in controlling lipid metabolism, which supports the ESC differentiation process and planarian organ maintenance (Deb et al., 2021). Another study found that TNFAIP2 overexpression enhanced TNT-mediated autophagosome and lysosome exchange, preventing advanced glycation end product (AGE)-induced autophagy and lysosome dysfunction and apoptosis (Barutta et al., 2023). In cancer treatment, TNFAIP2 was chosen as one of the six signature genes predicting chemotherapeutic and immunotherapeutic efficacies, with high-senescore patients benefiting from immunotherapy and low-senescore patients responsive to chemotherapy (Zhou et al., 2022).
These reports provide a possible explanation for previous studies showing that ITGB4 is important in EMT and cancer stemness. According to our results that there is an interaction between ITGB4 and TNFAIP2, ITGB4 might regulate EMT and stemness through TNFAIP2. TNFAIP2 is one of the important factors induced by tumor necrosis factor alpha (TNFα). Interestingly, TNFα release could be induced by therapeutic drugs from multiple tumor cell lines. The acquisition of docetaxel resistance was accompanied by increased constitutive production of TNFα (Guo and Yuan, 2020). In addition, TNFα is a key tumor-promoting effector molecule secreted by tumor-associated macrophages. In vitro neutralizing TNFα was observed to inhibit tumor progression and improve the curative effect of bevacizumab (Liu et al., 2020). Therefore, the mechanism by which TNFα promotes chemotherapeutic resistance in breast cancer should be further investigated.
For future studies, it will be important to develop Tnfaip2 knockout mice to investigate the exact role of TNFAIP2 physiologically. According to recent studies and our findings, agents targeting the interaction among ITGB4/TNFAIP2/IQGAP1 would be a promising trend for developing drugs to overcome the resistance phenomenon.
In summary, ITGB4 and TNFAIP2 play important roles in breast cancer chemoresistance. TNFAIP2 activates RAC1 to promote chemoresistance through IQGAP1. In addition, ITGB4 activates RAC1 through TNFAIP2 and IQGAP1 and confer DNA damage-related drug resistance in TNBC (Figure 8F). These results indicate that the ITGB4/TNFAIP2/IQGAP1/RAC1 axis provides potential therapeutic targets to overcome DNA damage-related drug resistance in TNBC.
All cell lines used in this study, including HCC1806, HCC1937, and HEK293T cells, were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA) and validated by STR (short tandem repeat) analysis and these cell lines tested negative for mycoplasma contamination. HCC1806 and HCC1937 cells were cultured in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS). HEK293T cells were cultured in DMEM (Thermo Fisher, Grand Island, USA) with 5% FBS at 37°C with 5% CO2. Epirubicin (EPI) (Cat#HY-13624A), cisplatin (DDP) (Cat#HY-17394), talazoparib (BMN) (Cat#HY-16106), and olaparib (AZD) (Cat#HY-10162) were purchased from MCE (New Jersey, USA).
We constructed the full-length TNFAIP2/ITGB4 gene and then subcloned them into the pCDH lentiviral vector. The packaging plasmids (including pMDLg/pRRE, pRSV-Rev, and pCMV-VSV-G) and pCDH-TNFAIP2/ITGB4 expression plasmid were cotransfected into HEK293T cells (2 × 106 in 10 cm plate) to produce lentivirus. Following transfection for 48 hr, the lentivirus was collected and used to infect HCC1806 and HCC1937 cells. Forty-eight hours later, puromycin (2 μg/ml) was used to screen the cell populations.
The pSIH1-H1-puro shRNA vector was used to express TNFAIP2, ITGB4, and luciferase (LUC) shRNAs. TNFAIP2shRNA#1, 5′-GACUUGGGCUCACAGAUAA-3′; TNFAIP2shRNA#2, 5′-GAUUGAGGUGGCCACUUAU-3′; ITGB4shRNA#1, 5′-ACGACAGCTTCCTTATGTA-3′; ITGB4shRNA#2, 5′-CAGCGACTACACTATTGGA-3′; LuciferaseshRNA, 5′-CUUACGCUGAGUACUUCGA-3′; HCC1806 and HCC1937 cells were infected with lentivirus. Stable populations were selected using 1–2 mg/ml puromycin. The knockdown effect was evaluated by WB.
The siRNA target sequences used in this study are as follows:TNFAIP2siRNA#1, 5′-GACUUGGGCUCACAGAUAA-3′; TNFAIP2siRNA#2, 5′-GAUUGAGGUGGCCACUUAU-3′; ITGB4siRNA#1, 5′-ACGACAGCTTCCTTATGTA-3′; ITGB4siRNA#2, 5′-CAGCGACTACACTATTGGA-3′; RAC1siRNA, 5′-CGGCACCACUGUCCCAACA-3′; IQGAP1siRNA#1, 5′-GCAGGTGGATTACTATAAA-3′; IQGAP1siRNA#2, 5′-CUAGUGAAACUGCAACAGA-3′. All siRNAs were synthesized by RiboBio (RiboBio, China) and transfected at a final concentration of 50 nM.
The WB procedure has been described in our previous study (Chen et al., 2005). Anti-TNFAIP2 (sc-28318), anti-ITGB4 (sc-9090), and anti-GAPDH (sc-25778) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-PARP (#9542) antibody was purchased from CST. Anti-RAC1 (05-389) and anti-γH2AX (3475627) antibodies were purchased from Millipore (Billerica, MA, USA). Anti-β-actin (A5441) and anti-Tubulin (T5168) antibodies were purchased from Sigma-Aldrich (St Louis, MO, USA). The anti-IQGAP1 (ab86064) antibody was purchased from Abcam.
Immunoprecipitation and silver staining lysates from HCC1806 cells stably expressing Flag-TNFAIP2 were prepared by incubating the cells in lysis buffer containing a protease inhibitor cocktail (MCE). Cell lysates were obtained from approximately 2.5 × 108 cells, and after binding with anti-Flag M2 affinity gel (Sigma) for 2 hr as recommended by the manufacturer, the affinity gel was washed with cold lysis plus 0.2% NP-40. FLAG peptide (Sigma) was applied to elute the Flag-labeled protein complex as described by the vendor. The elutes were collected and visualized on NuPAGE 4–12% Bis-Trisgels (Invitrogen, CA, USA) followed by silver staining with a silver staining kit (Pierce, IL, USA). The distinct protein bands were retrieved and analyzed by Liquid Chromatograph-Mass Spectrometer (LC‒mass).
For exogenous interaction between ITGB4 and Flag-TNFAIP2, cell lysates were directly incubated with anti-Flag M2 affinity gel (A2220; Sigm) overnight at 4°C. For endogenous protein interaction, cell lysates were first incubated with anti-TNFAIP2/ITGB4/IQGAP1 antibodies or mouse IgG/rabbit IgG (sc-2028; Santa Cruz Biotechnology, CA, USA) and then incubated with Protein A/G plusagarose beads (sc-2003; Santa Cruz Biotechnology). For the GST pull-down assay, cell lysates were directly incubated with GlutathioneSepharose 4B (52-2303-00; GE Healthcare) overnight at 4°C. The precipitates were washed four times with 1 ml of lysis buffer, boiled for 10 min with 1× sodium dodecyl sulfate (SDS) sample buffer, and subjected to WB analysis.
Cell viability was measured by SRB assays as described in our previous study (Chen et al., 2007). Cell viability was measured by SRB assays. Briefly, cells were seeded in 96-well plates. Then, the cells were cultured for the indicated time and fixed with 10% trichloroacetic acid at room temperature for 30 min, followed by incubation with 0.4% SRB (wt/vol) solution in 1% acetic acid for 20 min at room temperature. Finally, SRB was dissolved in 10 mM unbuffered Tris base, and the absorbance was measured at a wavelength of 530 nm on a plate reader (Bio Tek, Vermont, USA).
RAC1 activation was examined using the Cdc42 Activation Assay Biochem Kit (BK034, Cytoskeleton, Denver, USA) following the manufacturer’s instructions. Cells were harvested with cell lysis buffer, and1 mg of protein lysate in a 1 ml total volume at 4°C was immediately precipitated with 10 μg of PAK-PBD beads for 60 min with rotation. After washing three times with wash buffer, agarose beads were resuspended in 30 μl of 2× SDS sample buffer and boiled for 5 min. RAC1-GTP was examined by WB with an anti-RAC1 antibody.
We purchased 6- to 7-week-old female BALB/cnude mice from SLACCAS (Changsha, China). HCC1806-shLuc, HCC1806-shTNFAIP2, or HCC1806-shITGB4 cells (1 × 106 in Matrigel (BD Biosciences, NY, USA)) were implanted into the mammary fat pads of the mice. When the tumor volume reached approximately 50 mm3, the nude mice were randomly assigned to the control and treatment groups (n = 4/group). EPI, BMN, and DDP were dissolved in ddH2O. The control group was given vehicle alone, and the treatment group received EPI (2.5 mg/kg), BMN (1 mg/kg), and DDP (2.5 mg/kg) alone via intraperitoneal injection every 3 days for 18 or 27 days. The tumor volume was calculated as follows: tumor volume was calculated by the formula: (π × length × width2)/6.
Paraffin-embedded clinical TNBC specimens were obtained from the Department of Pathology, Henan Provincial People’s Hospital, Zhengzhou University, Henan, China. Two tissue microarrays containing 135 TNBC breast cancer tissues were constructed. For the IHC assay, the slides were deparaffinized, rehydrated, and pressure cooker heated for 2.5 min in EDTA for antigen retrieval. Endogenous peroxidase activity was inactivated by adding an endogenous peroxidase blocker (OriGene, China) for 15 min at room temperature. Slides were incubated overnight at 4°C with anti-TNFAIP2 (1:200) or anti-ITGB4 (1:500). After 12 hr, the slides were washed three times with PBS and incubated with secondary antibodies hypersensitive enzyme-labeled goat anti-mouse/rabbit IgG polymer (OriGene, China) at room temperature for 20 min, DAB concentrate chromogenic solution (1:200dilution of concentrated DAB chromogenic solution), counterstained with 0.5% hematoxylin, dehydrated with graded concentrations of ethanol for 3 min each (70–80–90–100%), and finally stained with dimethyl benzene immunostained slides were evaluated by light microscopy. The IHC signal was scored using the ‘Allred Score’ method.
All graphs were created using GraphPad Prism software version 8.0. Comparisons between two independent groups were assessed by two-tailed Student’s t-test. One-way analysis of variance with least significant differences was used for multiple group comparisons. p-values of <0.05, 0.01, or 0.001 were considered to indicate a statistically significant result, comparisons significant at the 0.05 level are indicated by *, at the 0.01 level are indicated by **, or at the 0.001 level are indicated by ***.
The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.
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In this study, Fang H et al. describe a potential pathway, ITGB4-TNFAIP2-IQGAP1-Rac1, that may involve in the drug resistance in triple negative breast cancer (TNBC). Mechanistically, it was demonstrated that TNFAIP2 bind with IQGAP1 and ITGB4 activating Rac1 and the following drug resistance. The present study focused on breast cancer cell lines with supporting data from mouse model and patient breast cancer tissues. The study is interesting. The experiments were well controlled and carefully carried out. The conclusion is supported by strong evidence provided in the manuscript. The authors may want to discuss the link between ITGB4 and Rac 1, between IQGAP1 and Rac1, and between TNFAIP2 and Rac1 as compared with the current results obtained. This is important considering some recent publications in this area (Cancer Sci 2021, J Biol Chem 2008, Cancer Res 2023). In addition, some key points need to be addressed in order to support their conclusion in full.https://doi.org/10.7554/eLife.88483.3.sa1
Breast cancer is the most common malignant tumor in women. One of subtypes in breast cancer is so called triple-negative breast cancer (TNBC), which represents the most difficult subtype to treat and cure in the clinic. Chemotherapy drugs including epirubicin and cisplatin are widely used for TNBC treatment. However, drug resistance remains as a challenge in the clinic. The authors uncovered a molecular pathway involved in chemotherapy drug resistance, and molecular players in this pathway represent as potential drug targets to overcome drug resistance. The experiments are well designed and the conclusions drawn mostly were supported by the data. The findings have potential to be translated into the clinic.https://doi.org/10.7554/eLife.88483.3.sa2
A summary of what the authors were trying to achieve:
- TNFAIP2 promotes TNBC drug resistance and DNA damage repair.
- Mechanistically, TNFAIP2 interacts with IQGAP1 and Integrin β4 to mediate RAC1 activation and thus promotes TNBC drug resistance.
- Clinically, TNFAIP2 expression levels positively correlated with ITGB4 in TNBC tissues.
- ITGB4 and TNFAIP2 might serve as promising therapeutic targets for TNBC.
-An account of the major strengths and weaknesses of the methods and results.
The authors performed numerous rescue experiments in vitro to confirm the relationship among ITGB4, TNFAIP2, IQGAP1 and Rac1. However, clinical relevance is somehow limited. Additional experiments are needed to demonstrate the above conclusions in clinical samples.
-An appraisal of whether the authors achieved their aims, and whether the results support their conclusions.
To most extent, the authors achieved their aims, and the results demonstrate their conclusions "TNFAIP2 interacts with IQGAP1 and ITGB4. ITGB4 promotes TNBC drug resistance via the TNFAIP2/IQGAP1/RAC1 axis by promoting DNA damage repair".
-A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community.
Drug resistance is always a challenge for TNBC treatment. This paper found that TNFAIP2 interacts with IQGAP1 and ITGB4 to activate Rac1, thus conferring DNA chemo-resistance to TNBC cells. In addition, positive correlation between the expression of TNFAIP2 and ITGB4 in TNBC tissues were presented. This paper suggests that the ITGB4/TNFAIP2/IQGAP1/Rac1 axis provides potential therapeutic targets to overcome chemo-resistance (DNA damage drugs) in fighting with TNBC.
Additional context to help readers interpret or understand the significance of the work:
This work reported a new mechanism related to TNBC chemo-resistance, which mainly depends on ordered interactions among ITGB4/TNFAIP2/IQGAP1/Rac1 and the following activation of pathways. Thus micro-peptide targeting technique, which is widely used to develop targeted drugs for protein-protein interactions, could show extraordinary potentials and application significance.
At present, cell penetrating peptide, a type of micro-peptide targeting technique, makes functional micro-peptides more stable by cross-linking some amino acid side chains. In recent years, it has been found that binding peptides can not only stabilize peptides, make them easier to enter cells, but also not easy to be hydrolyzed by proteases. At the same time, they have high affinity for targets and can target protein interactions, thus becoming a new way to develop protein interaction targeting inhibitors. To make it easier to enter cells, cell-penetrating peptides can be used in combination, such as HIV TAT. Cell-penetrating peptides can carry a variety of biologically active substances into the cell, is a good targeting drug carrier, with low toxicity, not limited by cell type, into the cell speed and high transduction efficiency. Based on the mechanism reported here, researchers can explore new micro-peptides targeting the interactions between ITGB4 and TNFAIP2 or TNFAIP2 and IQGAP1 to enhance the sensitivity of TNBC cells to drugs by cell-penetrating peptide technology.https://doi.org/10.7554/eLife.88483.3.sa3
The following is the authors’ response to the original reviews.
In this study, Fang H et al. describe a potential pathway, ITGB4-TNFAIP2-IQGAP1-Rac1, that may involve in the drug resistance in triple negative breast cancer (TNBC). Mechanistically, it was demonstrated that TNFAIP2 bind with IQGAP1 and ITGB4 activating Rac1 and the following drug resistance. The present study focused on breast cancer cell lines with supporting data from mouse model and patient breast cancer tissues. The study is interesting. The experiments were well controlled and carefully carried out. The conclusion is supported by strong evidence provided in the manuscript. The authors may want to discuss the link between ITGB4 and Rac1, between IQGAP1 and Rac1, and between TNFAIP2 and Rac1 as compared with the current results obtained. This is important considering some recent publications in this area (Cancer Sci 2021, J Biol Chem 2008, Cancer Res 2023). In addition, some key points need to be addressed in order to support their conclusion in full.
Thanks for your positive comments.
1. It is rarely found studies using the term of "DNA damage drug resistance". Do the authors mean "DNA damage and drug resistance" or "DNA damage-related drug resistance" or "DNA damage-induced drug resistance"? It is better to define "DNA damage drug resistance" in the manuscript if it is not a common term in the field.
We agree with you that the description "DNA damage-related drug resistance" is better so that we revised it uniformly in the manuscript.
2. For Figure 4A, it is stated the IQGAP1 is identified via IP-MS. However, the MS results are not presented in the Figure or in the supplementary. In Figure 4A, only the IP results with silver staining was presented. Moreover, based on the silver staining here, a bunch of proteins were increased in TNFAIP2 overexpression group compared to the vector group. Especially, there is a much clearer band at 52kDa. The authors didn't explain why they chose IQGAP1 and ITGB4 which are less clear than the protein(s) at 52kDa.
Supplementary file 1 is our mass spectrometry results. There are two reasons for choosing ITGB4 and IQGAP1. Firstly, we selected the proteins that indeed interact with TNFAIP2 according to our verification experiments. Secondly, we were interested in the mechanism by which TNFAIP2 promoting DNA damage-related drug resistance, and we found that ITGB4 promoted drug resistance, while IQGAP1 activated Rac1.
3. According to the images in Figure 4C, the efficiency of si-IQGAP1 is limited. The authors could analyze the WB image to confirm the inhibition efficiency of si-IQGAP1.
We analyzed the WB images and the quantitative results are as follows in Author response image 1. The knockdown efficiency is acceptable.
Author response image 1.
4. In Figure 5B, I wonder whether the authors can explain why the IgG could immunoprecipitate similar amount of ITGB4 protein as input group.
In this experiment, the Input group had relatively less loading amount (5%), while the IgG group had nonspecific binding.
5. According to the results from Figure 6B, the inhibition efficiency of shITGB4#1 is much higher than shITGB4#2. However, the effects of shITGB4#1 on GTP-Rac1 are similar to or even weaker than those of shITGB4#2 in both HCC1806 and HCC1937. Can this be explained?
The possible reason is that downregulation of ITGB4 expression to a certain level is sufficient to inhibit the activation of Rac1.
6. In Figure 6F, there are double bands for ITGB4 while only one band shows in other Figures. Please find a better representative image here.
ITGB4 has a cleaved band in addition to the main band. These two bands could be separated when we used a low concentration SDS-PAGE gel.
7. In the manuscript, GAPDH, b-Actin and Tubulin are used in different experiments as internal controls. Is there any specific reason to using different internal controls for different experiments here?
There is no specific reason using different internal controls. These experiments were conducted by different person. Each individual chose different internal controls based on the protein sizes.
8. I cannot find Table 1 for the correlation results for TNFAIP2 and ITGB4. I wonder whether Figure 8E is the Table 1 as is mentioned, since it is stated in line 561 that Figure 8E is "the work model of this paper" but actually Figure 8F is. If Figure 8E is the correlation results, I highly recommended the scatter plots graph is used here to present more clear and visualized correlation between TNFAIP2 and ITGB4.
Figure 8E is indeed the correlation result. In addition, Figure 8E could not be presented as scatter plot graph because the pattern of TNFAIP2 and ITGB4 expression is negative or positive according to the determination of IHC results which was carried out by professional pathologists.
9. Throughout the whole manuscript, no description of N number was found in figure legends or in Methods for in vitro experiments. N number is important for statistical analysis.
All our experiments have set up three replicates. We provide this information in figure legends.
Breast cancer is the most common malignant tumor in women. One of subtypes in breast cancer is so called triple-negative breast cancer (TNBC), which represents the most difficult subtype to treat and cure in the clinic. Chemotherapy drugs including epirubicin and cisplatin are widely used for TNBC treatment. However, drug resistance remains as a challenge in the clinic. The authors uncovered a molecular pathway involved in chemotherapy drug resistance, and molecular players in this pathway represent as potential drug targets to overcome drug resistance. The experiments are well designed and the conclusions drawn mostly were supported by the data. The findings have potential to be translated into the clinic.
Thanks for your positive comments.
1. In Introduction, the statement of "Breast cancer is the most common malignant tumor in women, and the morbidity and mortality rates of female malignant tumors are ranked first in the world" is inaccurate.
We have revised the description as“Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in women”.
2. In Materials and Methods, "Immunopurification and silver staining" is not correct, which should be replaced with "Immunoprecipitation and silver staining".
We replaced the description in the manuscript according to your suggestion.
3. It is unclear Why the authors chose the two TNBC cell lines, HCC1806 and HCC1937, for cell models in this work.
We chose these two cell lines according to our previous work“KLF5 promotes breast cancer proliferation, migration and invasion in part by upregulating the transcription of TNFAIP2” (doi: 10.1038/onc.2015.263. Epub 2015 Jul 20).
41. To demonstrate TNFAIP2 and ITGB4 confer TNBC drug resistance in vivo, the knockdown efficiency of animal experiments was not shown.
The knockdown efficiency of animal experiments was shown below. We added this result into Figure 2-figure supplement 2G and Figure 5-figure supplement 2N.
5. I would strongly suggest the authors seek help from a language editing service to improve the manuscript.
We improved the manuscript by using a professional English language editing service and we have carefully revised the manuscript.
In this manuscript, Fang and colleagues found that IQGAP1 interacts with TNFAIP2, which activates Rac1 to promote drug resistance in TNBC. Furthermore, they found that ITGB4 could interact with TNFAIP2 to promote TNBC drug resistance via the TNFAIP2/IQGAP1/Rac1 axis by promoting DNA damage repair.
This work has good innovation and high potential clinical significance. However, there are several unsolved concerns that have to be addressed.
Thanks for your positive comments.
1. In the manuscript, there are four drugs used for in vitro cell experiments, why is olaparib (AZD) not used for in vivo animal experiments？
There are two reasons why we did not choose AZD. First，the killing effect of AZD is not as strong as that of BMN. Second, AZD is more expensive than BMN. We finally chose BMN for animal experiments.
2. In Figure 4B, why the immunoprecipitation experiments is done in HCC1806 cell line？
In our previous study “KLF5 promotes breast cancer proliferation, migration and invasion in part by upregulating the transcription of TNFAIP2” (doi: 10.1038/onc.2015.263. Epub 2015 Jul 20), we found that TNFAIP2 knockdown could obviously inhibit the activation of Rac1 in HCC1806 when compared to the result in HCC1937. So, we used HCC1806 cell line to perform the IP-Mass assay.
3. There should be data showing the knockdown effect of TNFAIP2 and ITGB4 in animal experiments.
We addressed the same question above (Reviewer #2, Question#4).
4. When screening the interaction regions between ITGB4 and TNFAIP2, why the TNFAIP2 protein truncation strategy is to delete the N-terminus？
In fact, we also deleted the C-terminus, but the deletion of C-terminus of TNFAIP2 did not affect the interaction.
5. In the manuscript, "input" should be changed to "Input".
We corrected it.
6. There should be a space between "Figure" and numbers.
We add a space between "Figure" and numbers.https://doi.org/10.7554/eLife.88483.3.sa4
- Ceshi Chen
- Ceshi Chen
- Jing Feng
- Qiuxia Cui
- Huichun Liang
- Ceshi Chen
- Qiuxia Cui
- Ceshi Chen
- Ceshi Chen
The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.
This work was supported by National Key R&D Program of China (2020YFA0112300), National Natural Science Foundation of China (81830087, U2102203, 81672624, 82102987, and 82203413), the Yunnan Fundamental Research Projects (202101AS070050), the Guangdong Foundation Committee for Basic and Applied Basic Research projects (2022A1515012420), and Yunnan (Kunming) Academician Expert Workstation (grant no. YSZJGZZ-2020025).
Paraffin-embedded clinical TNBC specimens were obtained from the Department of Pathology, Henan Provincial People's Hospital, Zhengzhou University, Henan, China. This project has received medical ethics support (YS2021036).
Animal feeding and experiments were approved by the animal ethics committee of the affiliated Hospital of Guangdong Medical university (GDY2102096).
- Caigang Liu, Shengjing Hospital of China Medical University, China
- Yongliang Yang, Dalian University of Technology, China
You can cite all versions using the DOI https://doi.org/10.7554/eLife.88483. This DOI represents all versions, and will always resolve to the latest one.
© 2023, Fang, Ren, Cui 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.
Cancer stem cells (CSCs) undergo epithelial-mesenchymal transition (EMT) to drive metastatic dissemination in experimental cancer models. However, tumour cells undergoing EMT have not been observed disseminating into the tissue surrounding human tumour specimens, leaving the relevance to human cancer uncertain. We have previously identified both EpCAM and CD24 as CSC markers that, alongside the mesenchymal marker Vimentin, identify EMT CSCs in human oral cancer cell lines. This afforded the opportunity to investigate whether the combination of these three markers can identify disseminating EMT CSCs in actual human tumours. Examining disseminating tumour cells in over 12,000 imaging fields from 74 human oral tumours, we see a significant enrichment of EpCAM, CD24 and Vimentin co-stained cells disseminating beyond the tumour body in metastatic specimens. Through training an artificial neural network, these predict metastasis with high accuracy (cross-validated accuracy of 87-89%). In this study, we have observed single disseminating EMT CSCs in human oral cancer specimens, and these are highly predictive of metastatic disease.
Esophageal cancer (EC) is a fatal digestive disease with a poor prognosis and frequent lymphatic metastases. Nevertheless, reliable biomarkers for EC diagnosis are currently unavailable. Accordingly, we have performed a comparative proteomics analysis on cancer and paracancer tissue-derived exosomes from eight pairs of EC patients using label-free quantification proteomics profiling and have analyzed the differentially expressed proteins through bioinformatics. Furthermore, nano-flow cytometry (NanoFCM) was used to validate the candidate proteins from plasma-derived exosomes in 122 EC patients. Of the 803 differentially expressed proteins discovered in cancer and paracancer tissue-derived exosomes, 686 were up-regulated and 117 were down-regulated. Intercellular adhesion molecule-1 (CD54) was identified as an up-regulated candidate for further investigation, and its high expression in cancer tissues of EC patients was validated using immunohistochemistry, real-time quantitative PCR (RT-qPCR), and western blot analyses. In addition, plasma-derived exosome NanoFCM data from 122 EC patients concurred with our proteomic analysis. The receiver operating characteristic (ROC) analysis demonstrated that the AUC, sensitivity, and specificity values for CD54 were 0.702, 66.13%, and 71.31%, respectively, for EC diagnosis. Small interference (si)RNA was employed to silence the CD54 gene in EC cells. A series of assays, including cell counting kit-8, adhesion, wound healing, and Matrigel invasion, were performed to investigate EC viability, adhesive, migratory, and invasive abilities, respectively. The results showed that CD54 promoted EC proliferation, migration, and invasion. Collectively, tissue-derived exosomal proteomics strongly demonstrates that CD54 is a promising biomarker for EC diagnosis and a key molecule for EC development.