Introduction

Lung cancer is the leading cause of cancer-related deaths in both men and women with a 5-year survival rate of only 22% [1]. Although these outcomes may be improved by immune checkpoint blockade therapy involving the disruption of the binding of programmed cell death 1 (PD-1, also known as CD279) on tumor-infiltrating lymphocytes (TILs) to programmed death receptor ligand 1 (PD-L1, also known as B7-H1 or CD274) on tumor and tumor-associated cells, most lung cancer patients still fail the therapy, with a response rate of only about 20% [2]. In general, this revolutionary immunotherapy works better against ‘hot’ tumors, which have abundant TILs, strong immunogenicity and sufficient PD-L1 expression. Unfortunately, most tumors are ‘cold’, with low T-cell infiltration, weak immunogenicity and minimal PD-L1 expression, and show weak response to immune checkpoint inhibitors (ICIs) [3, 4].

Cold lung tumors without targetable oncogenic drivers are treated with chemotherapy as the standard approach [5]. However, the response rate to this conventional cancer therapy is also low and resistance often occurs after an initial response, with an overall survival (OS) of about 12-17 months. Given its roles in inducing TILs and immunogenicity, in particular PD-L1 expression, to turn cold tumors hot, chemotherapy can be an ideal candidate for combination with PD-1/PD-L1 blockade to improve therapeutic efficacy [6]. Indeed, combination treatment with ICIs and chemotherapeutic drugs shows synergy and better efficacy in both preclinical animal models and clinical trials of lung cancer and other cancers [6–8]. However, even with the combination of ICIs and chemotherapy, tumors in animals do not remit completely and the objective response rate (ORR) of lung cancer patients only reaches 33%-49.7%, with a median progression-free survival (PFS) of just 5.1-9 months and a median OS of 13-22 months [7, 8]. Thus, further improvement over the chemo-immunotherapy is direly needed.

Our recent human and mouse studies have shown that most lung tumors not only have low TILs and decreased PD-L1, but also down-regulate major histocompatibility complex class I (MHC-I), evading recognition and attack by CD8⁺ T cells, including those unleashed by ICIs and/or recruited by chemotherapy [6, 9, 10]. Following our previous cell line studies [11–17], we have established the PDZ-LIM domain-containing protein PDLIM2, also known as SLIM or mystique [18–20], as a bona fide tumor suppressor and its repression as a causative driver of lung cancer and resistance to ICIs and chemotherapeutic agents [6]. While PDLIM2 is epigenetically repressed in human lung cancer, associating with therapeutic resistance and poor prognosis, its global or lung epithelial-specific deletion in mice leads to lung cancer development, chemoresistance, and complete resistance to anti-PD-1 and epigenetic drugs. One most important function of PDLIM2 is to promote the ubiquitination and proteasomal degradation of nuclear signal transducer and activator of transcription 3 (STAT3) and nuclear factor-κB (NF-κB) RelA (also known as p65), two master transcription factors that function as proto-oncogenes in lung and many other cancers [6, 10–13, 21–27]. PDLIM2 repression in tumor cells thus results in the persistent activation of STAT3 and RelA, leading to MHC-I downregulation and high expression of tumor growth-related genes [6]. It also leads to strong induction of multi-drug resistance 1 (MDR1) for acquired chemo-resistance, as chemotherapy further enhances RelA activation for MDR1 transcription in PDLIM2 deficient tumor cells [6].

In this study, we examined whether and how PDLIM2 can be targeted to treat lung cancer in a faithful mouse model of human lung cancer. In particular, we tested whether systemic administration of nanoparticle-encapsulated PDLIM2-expression plasmids (nanoPDLIM2) could enhance the efficacy of anti-PD-1 and/or chemotherapeutic drugs. We also examined whether PDLIM2 repression in human lung cancer involves genetic deletion and its relationship with epigenetic silencing. These studies indicate that besides epigenetic repression, loss of heterozygosity (LOH) contributes to PDLIM2 downregulation in about 58% of human lung tumors, and that Pdlim2 heterozygous deletion (Pdlim2^+/-) mice develop spontaneous tumors in lung and other organs. Notably, systemic administration of nanoPDLIM2 reverses the phenotypes caused by PDLIM2 repression and induces complete remission of all lung tumors in most mice without further increasing toxicity when combined with anti-PD-1 and chemotherapeutic drugs. These findings provide a firm basis to combine ICIs and chemotherapeutic drugs with PDLIM2-targeted therapy for the treatment of lung and other cancers.

Material and Methods

Animals and lung carcinogenesis

We have complied with all relevant ethical regulations for animal testing and research. The animal experiments were performed in accordance with the US National Institutes of Health (NIH) Guidelines on the Use of Laboratory Animals. All animals were maintained under pathogen-free conditions and used according to protocols approved by the Institutional Animal Care and Use Committee (IACAUC) of the University of Pittsburgh. Pdlim2^flx/flx/SP-C-rtTA^tg/−/(tetO)7CMV-Cre^tg/tg (ΔSPC) mice under a pure FVB/N background and Pdlim2^-/- mice under a pure BALB/c background have been described before [6, 19, 21, 22, 28]. For lung carcinogenesis, six-to eight-week-old ΔSPC and wild type FVB/N mice were intraperitoneally (i.p.) injected with urethane (1 mg/g body weight, Sigma-Aldrich, St. Louis, MO, USA) once a week for six consecutive weeks [6], followed by different treatments as shown in the figures. Mice were euthanized at six weeks post urethane treatment for examination of lung tumors, inflammation, and treatment-induced toxicity. Surface tumors in mouse lungs were counted blinded under a dissecting microscope, and tumor diameters were measured by microcalipers.

Preparation of PDLIM2-expression plasmid or empty vector plasmid nanoparticles

The polyethylenimine (PEI)-based nanoparticles (in vivo-jetPEI) (Polyplus Transfection, New York, NY, USA) and plasmid DNA complexes at a nitrogen-to-phosphate ratio of 8 (N/P=8) were prepared according to the manufacturer’s instructions. Briefly, 25Lµg of pCMV-myc-Pdlim2 or empty vector plasmids in 100Lμl of a 5% glucose solution were mixed with the in vivo-jetPEI reagent (4Lμl) diluted into 100Lμl of a 5% glucose solution. After 15Lminutes of incubation at room temperature, the mixed solution (200 μl/mouse) was injected intravenously (i.v.) via the tail vein. PDLIM2-expression plasmid or empty vector plasmid DNA in tissues were measured by PCR assays targeting Amp-R in the genome of these plasmids.

Histology and immunohistochemistry (IHC) analysis

Lung, liver, kidney, and spleen tissues were excised, fixed in formalin, embedded in paraffin, and cut into 4-μm-thick sections. Sections were stained with H&E or subjected to sequential incubations with the indicated primary antibodies, biotinylated secondary antibodies and streptavidin-HRP [6]. Antibodies were listed in Supplementary Table S1.

In vivo BrdU labeling

Mice were i.p. injected with 50Lmg/kg BrdU (Sigma-Aldrich, St. Louis, MO, USA) 24Lh prior to euthanasia. Mouse lung tissue sections were stained with anti-BrdU (Sigma-Aldrich, St. Louis, MO, USA). BrdU labeling index was calculated as the percentage of labeled cells per total cells counted (> 500 cells in each counted tumor-containing area).

Flow cytometry (FACS) analysis

The cells were isolated from mouse lungs using collagenase/dispase digestion followed by filtration with 70 μm Nylon cell strainer and red blood cell lysis; then the cells were incubated with the antibodies against cell surface antigens after blocked with αCD16/CD32. The cells were then fixed with paraformaldehyde (2%), permeabilized and incubated with antibodies against intracellular antigens if needed. For interferon-γ (IFNγ) staining, cells were treated with phorbol 12-myristate 13-acetate (PMA, 50Lng/ml), ionomycin (1LμM), brefeldin A (BFA, 3Lμg/ml) and monensin (2LμM) for 4Lh before they were stained for FACS analysis. Data were acquired and analyzed by Accuri C6 or LSRFortessa I (BD Biosciences) and FlowJo software [29].

Quantitative polymerase chain reaction (qPCR) analysis

The indicated tissues or cells were subjected to DNA or RNA extraction, RNA reverse transcription and real-time PCR using trizol, reverse transcriptase, and Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA USA) according to manufacturer’s protocol. Primer pairs used for qPCR were listed in Supplementary Table S2.

Microsatellite and gene-specific PCR-based LOH analysis of PDLIM2

Genomic DNAs were isolated from human lung tumors and their matched normal tissues using the PureLink Genomic DNA Purification Kit (Invitrogen, Carlsbad, CA, USA), and subjected to semi-quantitative PCR using the primers specific for the microsatellite markers D8S1786 and D8S1752 that straddle the PDLIM2 genetic locus or the PDLIM2 genetic locus itself. Primer pairs used for the assays were listed in Supplementary Table S2.

Statistical analysis

One-way ANOVA power analysis was used to determine the minimum sample size. Animals were randomly assigned to different treatment groups. Measurements were taken from distinct samples. Student’s t test (two tailed) and one-way ANOVA/Tukey’s or two-way ANOVA/Sidak’s test were used to assess significance of differences between two groups and multiple comparisons, respectively. Pearson’s correlation test was used to assess association between PDLIM2 expression with its promoter methylation or genetic deletion and the overlap between PDLIM2 promoter methylation and genetic deletion. All bars in figures represent means ± standard error of the mean (SEM). The p values are indicated as *p < 0.05, **p < 0.01, ns, not statistically significant, except for those shown in figures. The p valuesL<L0.05 and 0.01 are considered statistically significant and highly statistically significant, respectively. Grubbs’ and ROUT outlier tests were established a priori. There were no exclusions in the analyses.

Results

Both epigenetic repression and genetic deletion of PDLIM2 in lung cancer

Using 40% of the expression level in matched normal lung tissues as the cut-off in the analysis of the Cancer Genome Atlas (TCGA) data, we found that PDLIM2 was repressed in over 75% of human lung tumors in a prior study [6]. If using 50% as the cut-off, PDLIM2 was repressed in about 93% of human lung tumors (Figure 1A). Using 125% of the methylation level of the PDLIM2 promoter in normal lung tissues as the cut-off to analyze TCGA database, over 70% of human lung tumors were found to have hypermethylation of the PDLIM2 promoter (Figure 1A, 1B), further validating our previous finding of epigenetic silencing as the main mechanism underlying PDLIM2 repression [6].

Figure 1.

In line with PDLIM2’s location on chromosome 8p21.3, a frequent LOH region in lung and other tumors [30–34], analysis of TCGA database revealed that PDLIM2 expression was positively associated with its gene copy numbers, and that over 58% of human lung tumors had genetic deletion of the PDLIM2 gene if copy number variation (CNV) of -0.1 was used as the cut-off for the gene deletion (Figure 1C). Further analysis indicated that about 44% of human lung tumors simultaneously harbored the promoter hypermethylation and LOH of the PDLIM2 gene, and approximately 26% and 14% of them only having the promoter hypermethylation or LOH of the PDLIM2 gene, respectively (Figure 1D). Around 16% of lung tumors possessed no such epigenetic or genetic alterations in PDLIM2. These findings were confirmed by microsatellite and gene-specific PCR-based LOH analysis of human primary lung tumor tissues and cell lines (Figure 1E, 1F; Supplementary Table S3). These data suggested that PDLIM2 repression in lung cancer involves both epigenetic silencing and genetic deletion.

To determine the significance of PDLIM2 LOH in lung tumors, we examined whether Pdlim2 heterozygous deletion leads to decreased PDLIM2 expression and spontaneous tumors in mice. Unlike its absolute absence in Pdlim2 homozygous deletion (Pdlim2^-/-) mice, PDLIM2 was detected in the lung of Pdlim2^+/- mice, but at a much lower level compared to wild type (WT) mice (Supplementary Figure S1A). Importantly, Pdlim2^+/- mice, like Pdlim2^-/- mice, also developed spontaneous tumors (Figure 1G). Of note, over 50% of tumors developed in Pdlim2^-/- and Pdlim2^+/- mice were lung tumors (Figure 1H; Supplementary Figure S1B). These data are also highly consistent with the fact that PDLIM2 is ubiquitously expressed under physiological conditions, with the highest level in the lung and lung epithelial cells in particular [6, 18–20]. Thus, PDLIM2 is a haploinsufficient tumor suppressor that is particularly important for lung tumor suppression.

Efficacy of systemic administration of nanoPDLIM2 in refractory lung cancer

Although reversal of PDLIM2 epigenetic repression by epigenetic drugs to restore PDLIM2 expression in cancer cells may be used to treat lung cancer [6], it is logical that this approach cannot be applied to lung tumors involving PDLIM2 LOH, which accounts for about 58% of all lung cancer cases. To overcome this limitation and expand PDLIM2-targeted therapy to all lung tumors with PDLIM2 repression regardless of the involved mechanisms, we tested the therapeutic efficacy of systemic administration of PDLIM2-expression plasmids encapsulated by the clinically feasible in vivo-jetPEI [35–39]. To this end, we employed mouse lung tumors induced by urethane, a faithful model of human lung cancer and adenocarcinoma (AC) in particular [6, 23, 24, 40–43]. Urethane is a chemical carcinogen present in fermented food, alcoholic beverage and also cigarette smoke, the predominant risk factor accounting for about 90% of human lung cancer cases [44]. Like its human counterpart, the murine lung cancer induced by urethane also shares PDLIM2 repression, in addition to their similarities in histology, genetics, molecular biology and immunology [6, 9, 23, 24, 40–43]. WT mice with lung tumors induced by urethane were i.v. injected with nanoparticle-encapsulated Pdlim2 plasmids or empty vector plasmids. Six weeks post the initial treatment of nanotherapy, mice were euthanized and lung tissues were collected (Figure 2A). Compared to the vector plasmid mock-treated group (Vec), nanoPDLIM2-treated mice had a significantly reduced tumor burden in their lungs, although the decrease in tumor numbers was not statistically significant (Figure 2B).

Figure 2.

In line with the tumor reduction, nanoPDLIM2 administration decreased nuclear RelA and STAT3, a hallmark of NF-κB and STAT3 activation, accordingly reduced the expression of their downstream cell survival gene Bcl-xL and cell proliferation gene Cyclin D1, increased apoptosis and decreased proliferation of lung cancer cells (Figure 2C-2E). These data indicated that intravenous administration of nanoPDLIM2 shows efficacy in suppressing oncogenic RelA and STAT3 activation and in treating lung cancer.

High tumor specificity and low toxicity of systemic administration of nanoPDLIM2

To characterize the potential new lung cancer therapy, we examined the expression levels of PDLIM2 in lung tumors and several organs, including the liver, kidney, and spleen. Consistent with the treatment efficacy, a high level of PDLIM2 was detected in the lung tumors from mice treated with the PDLIM2 plasmid nanoparticles one week post nanoPDLIM2 treatment, whereas no obvious PDLIM2 was found in the lung tumors from mice treated with the control plasmid nanoparticles (Figure 3A, 3B). It should be pointed out that the tumor delivery efficiency of PDLIM2-expression or control plasmid nanoparticles was similarly high (Figure 3B). However, either PDLIM2-expression or control plasmids were hardly detected in other organs/tissues of the same mice, including liver, kidney and spleen. Of note, in the first two days after injection, both empty vector and PDLIM2-expression plasmids were also detected at high levels in those tissues but quickly cleared afterward (data not shown), consistent with the well-documented tumor-specific enrichment of nanoparticles [37, 45–47]. Consistently, ectopic Pdlim2 was not detected, and the levels of PDLIM2 proteins were comparable in these tissues (Figure 3B, 3C).

Figure 3.

In further support of the lung tumor-specific delivery, the i.v. injection of either PDLIM2-expression or control plasmid nanoparticles showed no obvious toxicity to animals, as evidenced by no significant changes in the animal body weight and histology of all organs/tissues we examined, including spleen, kidney and liver (Figure 3D, E). Mouse appearance and behaviors, such as eating, drinking, defecating, urinating, sniffing, grooming, and digging, were not different between PDLIM2-expression and control plasmid nanoparticle groups (data not shown). Taken together, these data suggested the therapeutic efficacy and low toxicity of PDLIM2-based nanotherapy in the mouse model of refractory lung cancer.

Synergy of nanoPDLIM2 with chemotherapy in lung cancer treatment

Given the role of PDLIM2 in inhibiting the expression of cell survival and proliferation genes in tumor cells, which contribute to chemoresistance, we tested whether nanoPDLIM2 increases the efficacy of chemotherapy in the mouse model of lung cancer. Treatment with carboplatin and paclitaxel, two chemotherapeutic drugs that are often used together as the first-line treatment for lung and many other cancers, led to significant decrease in tumor number and tumor burden (Figure 4A). Importantly, combination with nanoPDLIM2 further significantly decreased both tumor number and tumor burden, suggesting a promising synergy between PDLIM2 nanotherapy and chemotherapy in lung cancer treatment. Consistently, significantly higher tumor cell apoptosis was detected in mice treated with the combination therapy, in comparison to those with nanoPDLIM2 or chemotherapy alone (Figure 4B).

Figure 4.

Another important mechanism contributing to the synergy between these two therapies involves nanoPDLIM2 blockade of the acquired chemoresistance of lung cancer cells. Chemotherapy induced strong RelA activation/nuclear expression and MDR1 expression (Figure 4C, 4D). NanoPDLIM2 not only repressed the constitutive activation of RelA in cancer cells but also prevented the strong induction of RelA activation and MDR1 expression by the chemotherapy. Thus, PDLIM2 nanotherapy improves the therapeutic efficacy of chemotherapy through blocking both intrinsic and acquired chemoresistance of lung cancer cells.

NanoPDLIM2 enhancement of ICI’s efficacy in lung cancer treatment

High expression of cell survival genes also renders tumor cells resistant to the tumoricidal activity of cytotoxic T lymphocytes (CTLs), including those unleashed by ICIs. PDLIM2 nanotherapy should also enhance the efficacy of immunotherapy, given its ability in suppressing the expression of cell survival genes. Furthermore, NanoPDLIM2 increased the number of TILs and the expression of MHC-I, the most important and core components of immunotherapies including PD-1 immune checkpoint blockade therapy (Figure 5A, 5B). In line with our previous studies [6], PD-1 blocking antibody showed some efficacies in the mouse model of lung cancer, as evidenced by the significant decrease in tumor burden and tumor number (Figure 5C). Combining with PDLIM2 nanotherapy further significantly decreased tumor burden, although the decrease in tumor number failed to reach statistical significance. Consistently, significant increases in both CD4⁺ and CD8⁺ TILs, CD8⁺ CTL activation and lung tumor cell death were detected (Figure 5D-5F).

Figure 5.

Complete remission of all lung tumors in most mice by the combination treatment of nanoPDLIM2 and anti-PD-1 and chemotherapeutic drugs

Like most human lung tumors, lung tumors in our animal model exhibit decreased expression of PD-L1 on cell surface, in additional to the low expression of MHC-I and low number of TILs [6, 9]. In line with our previous finding that PD-L1 expression is largely independent of PDLIM2 [6], nanoPDLIM2 failed to induce PD-L1 expression in lung tumors, which was in contrast to chemotherapy (Figure 6A). This may explain why the enhancing effect of nanoPDLIM2 on PD-1 blockade therapy is only moderate.

Figure 6.

Although it dramatically increases TILs as well [6], chemotherapy also induced the expression of PD-L1 on cancer cells (Figure 6A), which presumably protects cancer cells from immune attack and thereby restricts further efficacy improvement of its combination with PDLIM2 nanotherapy. But it may increase the sensitivity of cancer cells to PD-1 blockade therapy, particularly in combination with nanoPDLIM2. Indeed, chemotherapy and PD-1 blockade therapy showed a promising synergy in reducing both tumor number and tumor burden in comparison to the individual treatment of chemotherapy or PD-1 blockade therapy [6]. The synergy was largely blocked when Pdlim2 was genetically deleted from lung cancer cells (ΔSPC) (Figure 6B, 6C), suggesting an important role of PDLIM2 in the combination therapy of chemotherapeutic drugs and anti-PD-1. Whereas PDLIM2 nanotherapy increased MHC-I expression on tumor cells (Figure 5B), which is critical for better recognition and killing of tumor cells by CD8⁺ CTLs, chemotherapy alone or its combination with anti-PD-1 failed to do so (Figure 6D). Accordingly, combination of anti-PD-1 and chemotherapeutic drugs, like the combination therapies of nanoPDLIM2 and anti-PD-1 or chemotherapeutic drugs, also failed to induce a complete remission of lung tumors in any mice.

Given their overlapping roles in increasing TILs and in particular their complement roles in inducing MHC-I and PD-L1 expression on tumor cells, which turn tumors hot and more sensitive to PD-1 blockade, combination of nanoPDLIM2 and chemotherapeutic drugs with anti-PD-1 is expected to show better efficacy compared to combining only two of them. Indeed, combination of all three showed the strongest effect on reducing tumor burden and numbers, and caused complete remission of all lung tumors in 60% of mice (Figure 6E) and much more shrinkage of lung tumors in remaining mice in comparison to those treated with two combined therapies. In line with the high therapeutic efficacy, the triple combination therapy significantly increased the numbers and/or activation of CD4⁺ and CD8⁺ T cells in the lung, compared to the combination of chemotherapeutic drugs and anti-PD-1 (Figure 6F, 6G).

Of note, consistent with the undetectable toxicity of PDLIM2 nanotherapy, its co-treatment did not further increase the toxicity of anti-PD-1 and chemotherapeutic drugs. There was no obvious histological difference of major organs, including the liver, lung, kidney, and spleen (Supplementary Figure S2). Moreover, no significant differences in animal body weights were observed by additional nanoPDLIM2, in comparison to the mice received the combinational treatment of anti-PD-1 and chemotherapeutic drugs in the presence or absence of empty vector plasmid nanoparticles (Figure 6H). These data suggested a novel combination therapy with very high therapeutic efficacy and no increased toxicity for lung cancer, particularly refractory lung cancer.

Discussion

PD-1/PD-L1 blockade immunotherapy has recently joined chemotherapy as a standard treatment for lung and several other cancers [2–4]. While some patients have shown dramatic responses, most patients do not benefit from this novel treatment. Currently, various combinations of ICIs with other therapies, in particular its combination with chemotherapeutic drugs are being extensively tested in both preclinical and clinical trial studies to expand the benefit of this innovative therapy [6–8]. Although a promising synergy and better efficacy has been observed in both preclinical animal models and human clinical trial studies [6–8], significant further improvement is direly needed. Using an authentic mouse model of lung cancer, we show, for the first time, that PDLIM2 nanotherapy shows efficacy and high safety, and more importantly, induces complete remission of all lung tumors in most animals when it is combined with anti-PD-1 and chemotherapeutic drugs.

Most human lung tumors as well as lung tumors in our animal model have low numbers of TILs and decreased expression of PD-L1 and MHC-I on the cell surface [6, 9], all of which are important mechanisms contributing to the resistance to PD-1 blockade therapy. Through inducing immunogenic cell death (ICD) of cancer cells [8], chemotherapy can increase TILs and PD-L1 expression on tumor cells, and thereby synergize with anti-PD-1. However, chemotherapy cannot induce MHC-I expression, which limits further improvement of its synergy with immune therapy for complete cancer remission. Also, tumor cells usually express high levels of survival genes against the tumoricidal effects of chemotherapeutic drugs and of CTLs, including those activated by chemotherapy and unleashed by ICIs.

On the other hand, PDLIM2 nanotherapy induces MHC-I expression and lymphocyte tumor infiltration but does not up-regulate PD-L1. Moreover, PDLIM2 nanotherapy prevents the induction of MDR1 and the expression of tumor-related genes and in particular cell survival genes, further sensitizing tumor cells to the cytotoxicity of chemotherapeutic drugs and immune cells including those recruited by chemotherapy and unleashed by PD-1 blockade. Because of these important functions of PDLIM2 nanotherapy, PDLIM2 nanotherapy improved the efficacy of chemotherapy and PD-1 blockade therapy, and in combination with chemotherapy and PD-1 blockade therapy, resulted in complete cancer remission in most of the animals and dramatic tumor reduction in the remaining mice.

Another important clinical characteristic of PDLIM2 nanotherapy is its tumor-specificity and high safety profile. It delivered PDLIM2-expression plasmids to lung tumor tissues and showed undetectable toxicity in the animal model. Its combination does not further increase the toxicity of anti-PD-1 and chemotherapeutic drugs. This is in sharp contrast to the Food and Drug Administration (FDA)-approved epigenetic drugs, which can restore PDLIM2 expression in cancer cells with PDLIM2 epigenetic repression [6, 10–15]. Epigenetic drug treatment leads to body weight loss of animals (Supplementary Figure S3A). The toxicity is much worse when epigenetic drugs are combined with chemotherapy (Supplementary Figure S3).

Besides its side effects, epigenetic therapy should have much more limited application, even though it shows better efficacy as a monotherapy or combined with chemotherapeutic drugs or anti-PD-1 in comparison to PDLIM2 nanotherapy (Supplementary Figure S4). While nanoPDLIM2-based combination therapies could be applicable to all lung tumors with PDLIM2 repression regardless of the mechanisms involved, epigenetic therapy may be used to treat about 26% of lung tumors with PDLIM2 epigenetic repression only. About 58% of lung tumors harboring PDLIM2 LOH are not suitable to epigenetic therapies, although most of them are also with epigenetic alterations of the pdlim2 gene. Of note, the therapeutic efficacy of epigenetic therapy depends on PDLIM2 expression [6], and PDLIM2 heterozygous loss causes spontaneous lung and other cancers. Further, the efficacy and toxicity of the combination of epigenetic agents with both anti-PD-1 and chemotherapeutic drugs has not been examined yet.

Conclusions

In summary, the presented data identify genetic deletions as a major mechanism other than epigenetic alterations for PDLIM2 repression in human lung cancer, and PDLIM2 as a haploinsufficient tumor suppressor particularly important for suppressing lung cancer and therapy resistance. More importantly, these preclinical data establish a novel combination treatment of nanoPDLIM2, anti-PD-1 and chemotherapeutic drugs that induces complete remission of all lung tumors in most animals and is also with high safety profile. We believe that these knowledges are applicable to other cancers, because PDLIM2 repression has also been linked to numerous human cancers other than lung cancer.

Abbreviations

• AC: adenocarcinoma

• BFA: brefeldin A

• CNV: copy number variation

• CTL: cytotoxic T lymphocyte

• FACS: fluorescent activated cell sorting

• FDA: Food and Drug Administration

• IFNγ: interferon-γ

• ICD: immunogenic cell death

• ICI: immune checkpoint inhibitor

• IHC: immunohistochemistry

• i.p.: intraperitoneal

• i.v.: intravenous

• LOH: loss of heterozygosity

• MDR1: multi-drug resistance 1

• MHC-I: major histocompatibility complex class I

• nanoPDLIM2: nanoparticle-encapsulated PDLIM2-expression plasmids

• NF-κB: nuclear factor-κB

• NIH: National Institutes of Health

• ORR: objective response rate

• OS: overall survival

• PD-1: programmed cell death 1

• PD-L1: programmed death ligand 1

• PD-L2: programmed death ligand 2

• PDLIM2: PDZ-LIM domain-containing protein 2

• PEI: polyethylenimine

• PFS: progression-free survival

• PMA: phorbol 12-myristate 13-acetate

• qPCR: quantitative polymerase chain reaction

• SEM: standard error of the mean

• STAT3: signal transducer and activator of transcription 3

• TCGA: The Cancer Genome Atlas

• TIL: tumor-infiltrating lymphocyte

• WT: wild type.

Acknowledgements

The authors thank Dr. M. J. Grusby (Harvard School of Public Health) and Dr. J. A. Whitsett (University of Cincinnati College of Medicine) for providing PDLIM2^-/- and SP-C-rtTA^tg/−/(tetO)7CMV-Cre^tg/tg mice, respectively. The authors also thank Dr. W. Ma and Dr. S. Li (University of Pittsburgh) for their suggestions on the nanoparticle preparation, and Dr. L. H. Rigatti (University of Pittsburgh) for her histological diagnosis of mouse tissues.

Funding

This study was financially supported in part by the NIH National Institute of General Medical Sciences (NIGMS) grant R01 GM144890, National Cancer Institute (NCI) grants R01 CA172090 and R01 CA258614, R21 CA259706, American Cancer Society (ACS) Research Scholar grant RSG-19-166-01-TBG, and American Lung Association (ALA) Lung Cancer Discovery Award 821321.

Availability of data and materials

TCGA lung adenocarcinoma, lung squamous cell carcinoma, and lung cancer data we analyzed were obtained from https://portal.gdc.cancer.gov/projects. All data generated or analyzed during this study are included in this article and its additional files. Extra data that support the findings of this study are available from the corresponding authors upon reasonable request.

Author contributions

Z.Q. and G.X. conceived and designed the study, led and contributed to all aspects of the analysis, and wrote the manuscript. S.D.S. contributed to the overall experimental design and data analysis and interpretation, provided advice and constructive feedback, and edited the manuscript. F.S. performed and analyzed all the experimental assays. H.Z. contributed to data analysis and edited the manuscript. P.Y. originally identified PDLIM2 LOH in human lung tumors by gene-specific PCR-based LOH analysis. Y.X. contributed to mouse clone maintenance and IHC staining.

Ethics approval and consent to participate

All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh and carried out in accordance with NIH guidelines on animal care.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.