MIIP downregulates PD-L1 expression through HDAC6 in malignant melanoma

Abstract

Background Immune checkpoint inhibitors have improved the objective response rate and survival of melanoma patients. However, there are still many melanoma patients suffering from disease progression due to primary or secondary immune checkpoint inhibitor resistance, as is observed in the failure of anti-PD1/PD-L1 therapy. It is urgent to determine the function of PD-1/PD-L1 expression in melanoma and its associated pathways to enhance the efficacy of anti-PD-1 therapies.

Methods A cohort of 133 patients with histologically confirmed melanoma from Tianjin Medical University Cancer Institute & Hospital were included in this study. We performed immunohistochemical staining to detect the expression of MIIP, HDAC6 and PD-L1. Kaplan-Meier and log-rank test were used for survival analyze. As for vitro, Western blot was used to verify the signaling pathway that MIIP regulates PD-L1 expression.

Results Our present data demonstrate MIIP expression was decreased in melanoma and that the negative expression of MIIP was correlated with worse overall survival. We also found the positive expression of HDAC6, a molecule that is downstream of MIIP, had a positive trend with decreased overall survival, because the p value was not statistically significant. At the same time, the positive expression of PD-L1, a crucial costimulatory molecule, was associated with decreased overall survival. Furthermore, there was a positive association between HDAC6 and PD-L1 protein expression (p<0.01). In vitro experiment, we found that increasing MIIP led to decreased HDAC6, pSTAT3, and PD-L1 expression. Knocking down MIIP led to increased HDAC6, pSTAT3, and PD-L1 expression. Combining the published results, showing that HDAC6 can regulate PD-L1 expression through STAT3, our present data suggest that MIIP inhibits the expression of PD-L1 by downregulating HDAC6 in melanoma. Most importantly, methods for targeting MIIP-HDAC6-PD-L1 pathways, such as treatment with HDAC6 inhibitors, might indicate a new therapeutic approach for enhancing immune checkpoint inhibitor therapies in melanoma.

Conclusions Our findings highlight the immunomodulatory effects of MIIP in the inhibition of PD-L1 expression by downregulating HDAC6 in melanoma. Our data provide a sensible framework to consider the targeting of the MIIP-HDAC6-PD-L1 pathway.

Background

Malignant melanoma is a relatively common, aggressive tumor with an increasing incidence. It has a high mortality and a poor prognosis among patients with refractory and metastatic disease [1, 2]. In 2019, approximately 96,480 people were diagnosed with melanoma in the US, and estimated 7,230 patients died of this disease [3]. For patients with advanced and metastatic melanoma, the prognosis is still poor, The overall survival (OS) of 5 years is less than 10% with a median survival varying from 8 to 16 months [4]. Along with the rapid development of tumor immunology, treatment of melanoma by immunotherapy using the checkpoint inhibitors ipilimumab or the inhibitors of PD-1/PD-L1, e.g., nivolumab and pembrolizumab, shows great success in improving the prognosis for melanoma patients [5].

Immune evasion includes immune suppressive mechanism network in tumor microenvironment, which enables tumor cells to evade the recognition and attack of the immune system, thus allowing cell survival and proliferation [6]. CD8⁺ tumor infiltrating T lymphocytes (TILs) frequently express activated and produced inhibitory receptors, including PD-1 and CTLA-4 [7]. The immunosuppressive ligand PD-L1 (also known as B7-H1 or CD274) has been identified as a prognostic biomarker associated with poor survival and upregulated in many solid tumors [8]. The combination of PD-1 and PD-L1 deliver inhibitory signals that contribute to immune evasion, making this combination a potential immune checkpoint target [9]. In particular, several studies have indicated the effectiveness of anti-PD-1/PD-L1 treatment in patients with advanced malignant melanoma, which led PD-1/PD-L1 inhibitors to rapidly emerge as a common therapeutic option for advanced melanoma patients [10–12]. Treatment with immune checkpoint inhibitors of malignant melanoma patients exhibited a 5-year OS in 34% of the patients with a median OS of 23.8 months (95% CI, 20.2–30.4) and a 5-year PFS of in 21% of the patients with a median PFS of 8.3 months (95% CI, 5.8–11.1) [13]. However, immunotherapy is still largely ineffective in patients with tumors that have not been infiltrated by immune cells [13]. In addition, treatment-related AEs (TRAEs) occurred in more than 80% of melanoma patients [5]. Therefore, the discovery of new therapeutic methods and/or adjuvants that target multiple cellular processes is of special significance.

Migration and invasion inhibitory protein (MIIP), also known as invasion inhibitory protein 45 (IIp45) [14], was originally named for its ability to inhibit migration and invasion of tumor cells. It has recently been demonstrated to target downstream proteins to participate in tumor development and immune suppression [15–18]. In the yeast two-hybrid screen experiment to identify cellular regulators of insulin like growth factor binding protein 2 (IGFBP2), the promoter of human tumor cell migration, Song et al. first discovered MIIP gene and initially named IIp45 [14]. Furthermore, previous studies have demonstrated that MIIP can restrain the enzymatic activity of histone deacetylase 6 (HDAC6). Decreased acetylation activity of HDAC6 will result to α-tubulin acetylation and reduce cell migration [19]. In addition, MIIP was able to promote EGFR protein degradation and exert a negative effect on lung cancer cells’ proliferation [20].

As a main downstream target of MIIP and critical factor of histone acetylation and deacetylation, increased expression or the destroyed functional integrity of HDAC6 is inseparable with tumor [21–23]. It has been reported that HDAC6 is overexpressed in malignant melanoma, bladder cancer and lung cancer [2, 24]. In addition to being able to play the role of deacetylated histones, HDAC6 can also participate in the occurrence and development of tumors by targeting nonhistone substrates, such as heat shock protein 90 (HSP90), α-tubulin, and cortactin [24]. Interestingly, HDAC6 is also involved in the production of MHC class I proteins, specific tumor-associated antigens, cytokines, and the expression of costimulatory molecules [25]. Furthermore, melanoma antigens mart1, TYRP2, gp100, and TYRP1 were increased in mRNA expression levels after using HDAC6 inhibitors (Nexturastat A or Tubastatin A) [25]. Additionally, the major targets of cancer immunotherapy, programmed death receptor-1 (PD-1) and programmed death receptor ligand-1 (PD-L1) are also regulated by HDAC6 [4].

Although HDAC6 has been confirmed to be involved in the regulation of PD-L1 expression, the relationship between MIIP and the immune system is still unclear. Our present data suggest that MIIP inhibits the expression of PD-L1 by downregulating the expression of HDAC6 in melanoma. Methods of targeting MIIP-HDAC6-PD-L1 pathways, such as treatment with HDAC6 inhibitors, might be a new therapeutic approach for enhancing immune checkpoint inhibitor therapies in malignant melanoma. Thus, the already described participation of MIIP as an invasion-inhibitory protein and its new role as an upstream regulator of PD-L1 expression lays the foundation for its potential as a new treatment targeting MIIP-HDAC6-PD-L1 pathways.

Methods

Cell Lines, Cell Culture, Reagents, and siRNA Transfection

A875 and A375 melanoma cells (Beijing Cellular Research Institute, China) were cultured in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and the cells were incubated under 5% CO₂ at 37 °C. Overexpression of MIIP was achieved via transfection of Lipofectamine 3000 (Invitrogen) by following the manufacturer’s instructions with MIIP vector (GenePharma, Shanghai, China) for 48 hours. Depletion of MIIP in A375 and A875 cells was achieved by transfection with two different pools of siRNAs targeting MIIP (Thermo Fisher Scientific, ID: #1-123298 and #2-127111) using Lipofectamine RNAiMAX (Life Technologies, Grand Island, NY, USA); the transfection occurred over 24 h according to the manufacturer’s protocol.

Western Blot Analysis

After complete cell lysis, 30 µg of whole cell lysate was added to 10% polyacrylamide gels for electrophoresis. The cells were not treated with any cytokine. Primary antibodies for MIIP (#14472; Cell Signaling Technology, USA, 1:1000), HDAC6 (#13116; Cell Signaling Technology, USA, 1:1000), PD-L1 (#13684; Cell Signaling Technology, USA, 1:1000), and pSTAT3 (#8875; Cell Signaling Technology, USA, 1:1000) were used to analyze the expression of the proteins.

Results

Protein expression levels of MIIP, HDAC6 and PD-L1 and their correlations with clinicopathological parameters in malignant melanoma

The positive staining of MIIP and HDAC6 was mainly located in the cytoplasmic of cancer cells in melanoma tissues (Fig. 1a, b). PD-L1 was mainly expressed in the cytoplasmic and membrane of melanoma cells (Fig. 1c). Among the 133 malignant melanoma tissues, positive expression of MIIP could be detected in only 27.07% (36/133) patients. Positive expression of HDAC6 and PD-L1 was present in 61 (61/133, 45.86%) and 78 (78/133, 58.65%) cases, respectively.

We next investigated the associations of MIIP, HDAC6 and PD-L1 expression with clinicopathological parameters in malignant melanoma (Table 1). According to the results, the proportion of positive expression of PD-L1 was significantly higher in patients with at a higher Clark level [82.8% (53/64)] than it was in patients at a lower Clark level [56.5% (39/69)] (chi-square test, p < 0.01, Table 1). However, there were no statistically significant associations between MIIP, HDAC6 and PD-L1 expression level and patients’ genders, ages, serum LDH level, tumor size, or pathological TNM stages (chi-square test, p > 0.05, Table 1).

Melanoma patients with negative MIIP and positive PD-L1 expression have worse overall survival

Among the 133 malignant melanoma patients, the median follow-up time was 55.7 months (1.7–123.6 months), and there were 69 cancer-related deaths during the follow-up time. Kaplan-Meier analysis showed that OS in the positive MIIP expression group was longer than that in the negative MIIP expression group (p = 0.042) (Fig. 1d). In addition, patients with positive PD-L1 expression exhibited a shorter OS time than those with negative PD-L1 expression (p = 0.01) (Fig. 1f). A similar tendency was obtained for HDAC6, although statistical significance was not observed (p = 0.718) (Fig. 1e).

Correlation among MIIP, HDAC6 and PD-L1 expression

By analyzing the correlation between MIIP, HDAC6 and PD-L1 protein expression in Tissue Microarray (TMAs) constructed from 133 melanoma patient tissues, we demonstrated that HDAC6, a downstream molecule of MIIP, was positively associated with PD-L1 expression (p = 0.028, Table 2). Furthermore, we also found that when MIIP expression was positive, HDAC6 and PD-L1 were negative (Fig. 2a). Moreover, when MIIP expression was negative, HDAC6 and PD-L1 protein levels were positive (Fig. 2b). Thus, in malignant melanoma, there were positive trends in MIIP and HDAC6 protein expression as well as MIIP and PD-L1, although the statistical significance was not observed (p = 0.640, Table 3, p = 0.322, Table 4). Therefore, we speculated that in melanoma, MIIP might be involved in regulating PD-L1 expression through the MIIP-HDAC6-PD-L1 pathway.

MIIP regulates PD-L1 expression by downregulating HDAC6

To verify our speculation that MIIP regulates the expression of PD-L1 through HDAC6, the human malignant melanoma cell lines A875 and A375 were used as an in vitro model. For in vitro experiments, human melanoma cell lines were transfected with an MIIP overexpression vector. In melanoma cells that overexpressed MIIP, we observed a lower expression of HDAC6 and a decrease in phosphorylated STAT3 (Y705); we also observed a decrease in expression of PD-L1 (Fig. 3a). To ensure that off-target effect was not affecting our analysis, we used two different siRNAs to target MIIP for side-by-side experiments. The results showed that decreases in MIIP by siRNA treatment led to increased HDAC6 and pSTAT3 (Y705) activation, along with increased PD-L1 expression (Fig. 3b). These in vitro data provide further evidence that MIIP downregulates PD-L1 expression through HDAC6 in malignant melanoma. Thus, based on in vivo and in vitro data, we suggest that in malignant melanoma, MIIP downregulates PD-L1 expression through HDAC6 (Fig. 4).

Discussion

Anti-PD-1 treatment have achieved advanced success in malignant melanoma. While the expression of valuable markers, such as TMB, MSI, and PD-L1, could serve as effective predictors of anti-checkpoint inhibitor therapies, not all melanoma patients are effective to this treatment. In a recent study, the objective response rate (ORR) is 41% in all melanoma patients and 52% for treatment-naive melanoma patients [5]. Additionally, treatment-related AEs (TRAEs) occurred in 86% of patients and resulted in study discontinuation in 7.8% of patients; 17% experienced grade 3/4 TRAE [5]. Although immunotherapy has greatly improved the prognosis of patients, immunotherapy remains largely ineffective in patients with tumors not infiltrated by immune cells. In order to incrementally advance the immunotherapeutic options in melanoma treatment, the discovery of new therapeutic methods and/or adjuvants that target multiple cellular processes shows special significance.

Previous studies have reported that MIIP plays a role as a tumor suppressor gene. In gliomas, through binding to HDAC6, highly expressed MIIP causes decreased HDAC6 expression and inhibition of HDAC6 deacetylase activity, thereby inhibiting HDAC6-mediated cell migration [19]. The tumor suppressor function of MIIP has been demonstrated in tissues from breast cancer [30] and colorectal cancer [31]. In the current study, we verified that melanoma patients exhibited a negative MIIP expression and predicted worse overall survival when compared with patients with a positive MIIP expression. We also found the positive expression of HDAC6, a molecule that is downstream of MIIP, had a positive trend with decreased overall survival, because the p value was not statistically significant. The reason for this inconsistency may be due to our insufficient sample size and rough melanoma subtype classification. We will collect more samples and perform more precise subtype classification to explore the role of HDAC6 in melanoma. At the same time, the positive expression of PD-L1, an important costimulatory molecule expressed in cancer cells, was associated with worse overall survival. Furthermore, there was a positive association between HDAC6 and PD-L1.

When we studied the relationship between the expression levels of MIIP, HDAC6 and PD-L1 and the clinicopathological factors in melanoma patients, a correlation between PD-L1 and melanoma cell Clark stage was identified. The expression rate of PD-L1 was significantly greater in higher Clark levels [82.8% (53/64)] than it was in lower Clark levels [56.5% (39/69)] (chi-square test, p < 0.01). The Clark stage classification is defined by measuring the depth of skin invasion of melanoma cell to the anatomical level. And it provides a correlation between the degree of skin invasion by melanoma and the 5-year survival rate after surgery. In malignant melanoma, the relationship between PD-L1 expression and Clark stage may exhibit a greater likelihood of malignant behaviors. However, this needs further study for validation.

Some HDACs have received particular attention for their recently endowed roles in regulating tumorigenesis and immune response [32, 33]. However, HDACi's ability to regulate cellular immune microenvironment and their therapeutic potential as targeted agents combined with immunotherapy are not clear. Histone deacetylases (HDACs) and selective HDAC inhibitors (HDACi), alone or in combination with other anti-cancer agents, are promising therapeutic methods in many cancers [34–37]. As a major transcription factor regulating PD-L1, Lienlaf et al demonstrated that HDAC6 was crucial adjective for the recruitment and activation of STAT3 and the upregulation of PD-L1. Additionally, a study has demonstrated that in the mouse model of B16F10 immunotherapy, HDACi combined with PD-1/PD-L1 checkpoint inhibitors can significantly improve the therapeutic effect of immunotherapy alone [38] In multiple myeloma, the expression of PD-L1 is immediately correlated with disease progression. By contrast, the highest PD-L1 expression was observed in patients with recurrent / refractory multiple myeloma.[39] ACY-241, the HDAC6 selective inhibitor, combined treatment with anti-PD-L1 treatment can enhance anti-multiple myeloma immunity in the bone marrow microenvironment through down-regulating the interaction between pDC-T cell and pDC-NK cell.[36] A recent study also showed that a HDAC6i, ricolinostat, promoted phenotypic changes that supported the activation of T cells and improved the function of antigen presenting cells.[40] Furthermore, The use of histone deacetylase 6 (HDAC6) inhibitors limited the growth of ovarian cancer with mutations in ARID1A, which is the most common mutations in human cancer epigenetic regulation factor; Mutations in ARID1A occur in more than 50% of ovarian clear cell carcinomas and it modulates the tumor immune microenvironment.[41] Regarding our present study which suggests that MIIP inhibits the expression of PD-L1 by downregulating the expression of HDAC6 in melanoma, methods that target MIIP-HDAC6-PD-L1 pathways, such as treatment with HDAC6, might provide a new therapeutic approach to enhance immune checkpoint inhibitor therapies in malignant melanoma.

In addition to HDAC6, MIIP can also regulate the expression of insulin-like growth factor-binding protein 2 (IGFBP2), and it can accelerate epidermal growth factor receptor (EGFR) protein turnover and attenuate proliferation [14, 20]. Accumulating evidence has suggested that IGFBP2 modulates the immune response in cancer patients and can be a potential target for cancer immunotherapy [42]. Although an IGFBP2 vaccine was shown to be immunosuppressive, removing the IL-10-inducing T helper epitopes from the vaccine was suggested to ensure potent IGFBP2 anti-tumor activity [43]. Furthermore, many clinical trials have investigated EGFR-mediated tumor immune escape as a target for immunotherapy through the use of immune checkpoint inhibitors. Concha-Benavente et al. (2013) found that overexpression of EGFR in response to IFN-γ through the JAK2/STAT1 pathway upregulated PD-L1 expression and that specific inhibition of JAK2 abolished PD-L1 upregulation in head and neck cancer. In another study, the mutated and constitutively active EGFR/KRAS-MAPK pathway was suggested to cause upregulation of PD-L1 in non-small-cell lung cancer [44]. However, as an upstream gene of IGFBP2, HDAC6 and EGFR, the mechanism of MIIP involvement in immune regulation requires further investigation.

Conclusion

Our findings highlight the immunomodulatory effects of MIIP, which include the inhibition of the expression of PD-L1 by downregulating the expression of HDAC6 in melanoma. Our data provide a sensible framework to consider the targeting of the MIIP-HDAC6-PD-L1 pathway. For example, HDAC6 might indicate a new therapeutic approach to enhance immune checkpoint inhibitor therapies in malignant melanoma.

Abbreviations

PD-1: programmed cell death 1; PD-L1; programmed cell death-ligand 1; STAT3: signal transducer and activator of transcription 3; MIIP: migration and invasion inhibitory protein; HDAC6: Histone deacetylase 6; DMEM: Dulbecco-modified essential medium; FBS: fetal bovine serum; OS: overall survival.

Declarations

Ethics approval and consent to participate

The present study was approved by the Institutional Ethics Committee of Tianjin Cancer Hospital. All patients signed written informed consent forms.

Consent to publish

Not applicable.

Availability of data and materials

The datasets generated and/or analyzed during the current study are not publicly available due to data privacy according to the license for the current study but are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was funded by Key Nature Science Foundation of Tianjin (18YFZCSY00550 to J. Yang). Jilong Yang is the undertaker of the fund.

Authors’ Contributions

JLY designed the study and provided funding for the study. RWX and TL participated in the entire experiment and writing and modifying the manuscript. HTL, JQW, JL participated in the editing and revision of the manuscript. LJX participated in the statistical analysis of the data. All authors read and approved the manuscript.

Acknowledgments

This research was funded by Key Nature Science Foundation of Tianjin (18YFZCSY00550 to J. Yang). We thank the American Journal Experts (https://www.aje.cn/) for the help with the English editing. We thank the reviewers and editors for their constructive comments.

References

1. Hao MZ, Zhou WY, Du XL, Chen KX, Wang GW, Yang Y, Yang JL. Novel anti-melanoma treatment: focus on immunotherapy. Chinese journal of cancer. 2014;33(9):458–65.
2. Hao M, Song F, Du X, Wang G, Yang Y, Chen K, Yang J. Advances in targeted therapy for unresectable melanoma: new drugs and combinations. Cancer letters. 2015;359(1):1–8.
3. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA: a cancer journal for clinicians 2018, 68(1):7–30.
4. Lienlaf M, Perez-Villarroel P, Knox T, Pabon M, Sahakian E, Powers J, Woan KV, Lee C, Cheng F, Deng S, et al: Essential role of HDAC6 in the regulation of PD-L1 in melanoma. Molecular oncology 2016.
5. Hamid O, Robert C, Daud A, Hodi FS, Hwu WJ, Kefford R, Wolchok JD, Hersey P, Joseph R, Weber JS, et al. Five-year survival outcomes for patients with advanced melanoma treated with pembrolizumab in KEYNOTE-001. Annals of oncology: official journal of the European Society for Medical Oncology / ESMO. 2019;30(4):582–8.
6. Ko JS. The Immunology of Melanoma. Clinics in laboratory medicine. 2017;37(3):449–71.
7. Wei SC, Levine JH, Cogdill AP, Zhao Y, Anang N-AAS, Andrews MC, Sharma P, Wang J, Wargo JA, Pe'er D, et al. Distinct Cellular Mechanisms Underlie Anti-CTLA-4 and Anti-PD-1 Checkpoint Blockade. Cell. 2017;170(6):1120–33.e1117.
8. Daud AI, Wolchok JD, Robert C, Hwu WJ, Weber JS, Ribas A, Hodi FS, Joshua AM, Kefford R, Hersey P, et al. Programmed Death-Ligand 1 Expression and Response to the Anti-Programmed Death 1 Antibody Pembrolizumab in Melanoma. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2016;34(34):4102–9.
9. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, Drake CG, Camacho LH, Kauh J, Odunsi K, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012;366(26):2455–65.
10. Hamid O, Robert C, Daud A, Hodi FS, Hwu W-J, Kefford R, Wolchok JD, Hersey P, Joseph RW, Weber JS, et al. Safety and Tumor Responses with Lambrolizumab (Anti–PD-1) in Melanoma. N Engl J Med. 2013;369(2):134–44.
11. Goldberg SB, Gettinger SN, Mahajan A, Chiang AC, Herbst RS, Sznol M, Tsiouris AJ, Cohen J, Vortmeyer A, Jilaveanu L, et al. Pembrolizumab for patients with melanoma or non-small-cell lung cancer and untreated brain metastases: early analysis of a non-randomised, open-label, phase 2 trial. The Lancet Oncology. 2016;17(7):976–83.
12. Ott PA, Hu Z, Keskin DB, Shukla SA, Sun J, Bozym DJ, Zhang W, Luoma A, Giobbie-Hurder A, Peter L, et al. An Immunogenic Personal Neoantigen Vaccine for Melanoma Patients. Nature. 2017;547(7662):217–21.
13. Heinhuis KM, Ros W, Kok M, Steeghs N, Beijnen JH, Schellens JHM. Enhancing antitumor response by combining immune checkpoint inhibitors with chemotherapy in solid tumors. Ann Oncol. 2019;30(2):219–35.
14. Song SW, Fuller GN, Khan A, Kong S, Shen W, Taylor E, Ramdas L, Lang FF, Zhang W. IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion. Proc Natl Acad Sci USA. 2003;100(24):13970–5.
15. Wang Y, Hu L, Ji P, Teng F, Tian W, Liu Y, Cogdell D, Liu J, Sood AK, Broaddus R, et al. MIIP remodels Rac1-mediated cytoskeleton structure in suppression of endometrial cancer metastasis. J Hematol Oncol. 2016;9(1):112.
16. Chen T, Li J, Xu M, Zhao Q, Hou Y, Yao L, Zhong Y, Chou P-C, Zhang W, Zhou P, et al. PKCε phosphorylates MIIP and promotes colorectal cancer metastasis through inhibition of RelA deacetylation. Nature communications. 2017;8(1):939–9.
17. Du Y, Wang P. Upregulation of MIIP regulates human breast cancer proliferation, invasion and migration by mediated by IGFBP2. Pathology - Research Practice. 2019;215(7):152440.
18. Yan G, Ru Y, Yan F, Xiong X, Hu W, Pan T, Sun J, Zhang C, Wang Q, Li X. MIIP inhibits the growth of prostate cancer via interaction with PP1α and negative modulation of AKT signaling. Cell Commun Signal. 2019;17(1):44–4.
19. Wu Y, Song SW, Sun J, Bruner JM, Fuller GN, Zhang W. IIp45 inhibits cell migration through inhibition of HDAC6. J Biol Chem. 2010;285(6):3554–60.
20. Wen J, Fu J, Ling Y, Zhang W. MIIP accelerates epidermal growth factor receptor protein turnover and attenuates proliferation in non-small cell lung cancer. Oncotarget. 2016;7(8):9118–34.
21. Zhang Z, Yamashita H, Toyama T, Sugiura H, Omoto Y, Ando Y, Mita K, Hamaguchi M, Hayashi S-i, Iwase H. HDAC6 Expression Is Correlated with Better Survival in Breast Cancer. Clin Cancer Res. 2004;10(20):6962.
22. Saji S, Kawakami M, Hayashi S-i, Yoshida N, Hirose M, Horiguchi S-i, Itoh A, Funata N, Schreiber SL, Yoshida M, et al. Significance of HDAC6 regulation via estrogen signaling for cell motility and prognosis in estrogen receptor-positive breast cancer. Oncogene. 2005;24(28):4531–9.
23. Bazzaro M, Lin Z, Santillan A, Lee MK, Wang MC, Chan KC, Bristow RE, Mazitschek R, Bradner J, Roden RB. Ubiquitin proteasome system stress underlies synergistic killing of ovarian cancer cells by bortezomib and a novel HDAC6 inhibitor. Clinical cancer research: an official journal of the American Association for Cancer Research. 2008;14(22):7340–7.
24. Li T, Zhang C, Hassan S, Liu X, Song F, Chen K, Zhang W, Yang J. Histone deacetylase 6 in cancer. J Hematol Oncol. 2018;11(1):111–1.
25. Woan KV, Lienlaf M, Perez-Villaroel P, Lee C, Cheng F, Knox T, Woods DM, Barrios K, Powers J, Sahakian E, et al. Targeting histone deacetylase 6 mediates a dual anti-melanoma effect: Enhanced antitumor immunity and impaired cell proliferation. Molecular oncology. 2015;9(7):1447–57.
26. Wen J, Liu QW, Luo KJ, Ling YH, Xie XY, Yang H, Hu Y, Fu JH. MIIP expression predicts outcomes of surgically resected esophageal squamous cell carcinomas. Tumour biology: the journal of the International Society for Oncodevelopmental Biology and Medicine 2016.
27. Crane CA, Panner A, Murray JC, Wilson SP, Xu H, Chen L, Simko JP, Waldman FM, Pieper RO, Parsa AT. PI(3) kinase is associated with a mechanism of immunoresistance in breast and prostate cancer. Oncogene. 2009;28(2):306–12.
28. Wang L, Xiang S, Williams KA, Dong H, Bai W, Nicosia SV, Khochbin S, Bepler G, Zhang X. Depletion of HDAC6 enhances cisplatin-induced DNA damage and apoptosis in non-small cell lung cancer cells. PloS one. 2012;7(9):e44265–5.
29. Zhang M, Wang D, Sun Q, Pu H, Wang Y, Zhao S, Wang Y, Zhang Q. Prognostic significance of PD-L1 expression and (18)F-FDG PET/CT in surgical pulmonary squamous cell carcinoma. Oncotarget. 2017;8(31):51630–40.
30. Song F, Ji P, Zheng H, Song F, Wang Y, Hao X, Wei Q, Zhang W, Chen K. Definition of a functional single nucleotide polymorphism in the cell migration inhibitory gene MIIP that affects the risk of breast cancer. Cancer research. 2010;70(3):1024–32.
31. Sun Y, Ji P, Chen T, Zhou X, Yang D, Guo Y, Liu Y, Hu L, Xia D, Liu Y, et al: MIIP haploinsufficiency induces chromosomal instability and promotes tumour progression in colorectal cancer. The Journal of pathology 2016.
32. Woan KV, Sahakian E, Sotomayor EM, Seto E, Villagra A. Modulation of antigen-presenting cells by HDAC inhibitors: implications in autoimmunity and cancer. Immunol Cell Biol. 2012;90(1):55–65.
33. Kroesen M, Gielen P, Brok IC, Armandari I, Hoogerbrugge PM, Adema GJ. HDAC inhibitors and immunotherapy; a double edged sword? Oncotarget 2014, 5(16):6558–6572.
34. Amengual JE, Johannet P, Lombardo M, Zullo K, Hoehn D, Bhagat G, Scotto L, Jirau-Serrano X, Radeski D, Heinen J, et al. Dual Targeting of Protein Degradation Pathways with the Selective HDAC6 Inhibitor ACY-1215 and Bortezomib Is Synergistic in Lymphoma. Clinical cancer research: an official journal of the American Association for Cancer Research. 2015;21(20):4663–75.
35. Suraweera A, O'Byrne KJ, Richard DJ. Combination Therapy With Histone Deacetylase Inhibitors (HDACi) for the Treatment of Cancer: Achieving the Full Therapeutic Potential of HDACi. Frontiers in oncology. 2018;8:92–2.
36. Ray A, Das DS, Song Y, Hideshima T, Tai YT, Chauhan D, Anderson KC. Combination of a novel HDAC6 inhibitor ACY-241 and anti-PD-L1 antibody enhances anti-tumor immunity and cytotoxicity in multiple myeloma. Leukemia. 2018;32(3):843–6.
37. Knox T, Sahakian E, Banik D, Hadley M, Palmer E, Noonepalle S, Kim J, Powers J, Gracia-Hernandez M, Oliveira V, et al. Selective HDAC6 inhibitors improve anti-PD-1 immune checkpoint blockade therapy by decreasing the anti-inflammatory phenotype of macrophages and down-regulation of immunosuppressive proteins in tumor cells. Scientific reports. 2019;9(1):6136.
38. Woods DM, Sodré AL, Villagra A, Sarnaik A, Sotomayor EM, Weber J. HDAC Inhibition Upregulates PD-1 Ligands in Melanoma and Augments Immunotherapy with PD-1 Blockade. Cancer immunology research. 2015;3(12):1375–85.
39. Görgün G, Samur MK, Cowens KB, Paula S, Bianchi G, Anderson JE, White RE, Singh A, Ohguchi H, Suzuki R, et al. Lenalidomide Enhances Immune Checkpoint Blockade-Induced Immune Response in Multiple Myeloma. Clinical cancer research: an official journal of the American Association for Cancer Research. 2015;21(20):4607–18.
40. Adeegbe DO, Liu Y, Lizotte PH, Kamihara Y, Aref AR, Almonte C, Dries R, Li Y, Liu S, Wang X, et al. Synergistic Immunostimulatory Effects and Therapeutic Benefit of Combined Histone Deacetylase and Bromodomain Inhibition in Non-Small Cell Lung Cancer. Cancer discovery. 2017;7(8):852–67.
41. Fukumoto T, Fatkhutdinov N, Zundell JA, Tcyganov EN, Nacarelli T, Karakashev S, Wu S, Liu Q, Gabrilovich DI, Zhang R. HDAC6 Inhibition Synergizes with Anti-PD-L1 Therapy in ARID1A-Inactivated Ovarian Cancer. Cancer research. 2019;79(21):5482.
42. Patil SS, Railkar R, Swain M, Atreya HS, Dighe RR, Kondaiah P. Novel anti IGFBP2 single chain variable fragment inhibits glioma cell migration and invasion. Journal of Neuro-Oncology. 2015;123(2):225–35.
43. Cecil DL, Holt GE, Park KH, Gad E, Rastetter L, Childs J, Higgins D, Disis ML. Elimination of IL-10 inducing T-helper epitopes from an IGFBP-2 vaccine ensures potent anti-tumor activity. Cancer research. 2014;74(10):2710–8.
44. Akbay EA, Koyama S, Carretero J, Altabef A, Tchaicha JH, Christensen CL, Mikse OR, Cherniack AD, Beauchamp EM, Pugh TJ, et al: Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors. Cancer discovery 2013, 3(12):10.1158/2159–8290.CD-1113-0310.