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Many cancers carry recurrent, change-of-function mutations affecting RNA splicing factors, however, less is known about the consequences of upregulated RNA splicing factors (SFs) in cancer. Here, we describe a role of survival motor neuron domain containing 1 (SMNDC1) protein, a poorly studied SF that we found to be upregulated in pancreatic ductal adenocarcinoma (PDAC) and high-grade serous ovarian cancers (HGSOCs) and associated with poor patient prognosis. By loss- and gain-of- function approaches we found that SMNDC1 promotes cancer cell proliferation, clonal expansion and tumor growth. Mechanistically, SMNDC1 promoted the retention of A/C-rich exons which otherwise would be excluded or less retained after RNA splicing in normal cells. Inclusion of exon 4 (E4) of MAPK3 (ERK1), which encodes both phosphorylation sites (Thr202/Tyr204) of the kinase, was among the promoted exons by SMNDC1. Forced exclusion of MAPK3 E4 using anti-sense oligos in vitro and in vivo, inhibited the phosphorylation of ERK1/2, the expression of downstream genes (Cyclin D1, Elk1) and increased proapoptotic proteins. These data provide a novel mechanism by which PDAC and HGSOC cells exploit a “splicing switch” that increases ERK1 phosphorylation, and offer a druggable alternative to target these cancer cells by jointly blocking oncogenic signaling and altered RNA splicing. Citation Format: Yushan Zhang, Md Afjalus Siraj, Prabir Chakrabarty, Robert Tseng, Shamik Das, Resham Bhattacharya, Luisa Escobar-Hoyos, Priyabrata Mukherjee. Activation of the MAPK pathway by altered RNA splicing in cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B062.

G protein–coupled receptor kinases (GRK) regulate diverse cellular functions ranging from metabolism to growth and locomotion. Here, we report an important contributory role for GRK5 in human prostate cancer. Inhibition of GRK5 kinase activity attenuated the migration and invasion of prostate cancer cells and, concordantly, increased cell attachment and focal adhesion formation. Mass spectrometric analysis of the phosphoproteome revealed the cytoskeletal-membrane attachment protein moesin as a putative GRK5 substrate. GRK5 regulated the subcellular distribution of moesin and colocalized with moesin at the cell periphery. We identified amino acid T66 of moesin as a principal GRK5 phosphorylation site and showed that enforcing the expression of a T66-mutated moesin reduced cell spreading. In a xenograft model of human prostate cancer, GRK5 silencing reduced tumor growth, invasion, and metastasis. Taken together, our results established GRK5 as a key contributor to the growth and metastasis of prostate cancer. Cancer Res; 74(13); 3489–500. ©2014 AACR.

Androgen receptor (AR) is an androgen-activated transcription factor and drives prostate cancer (PCa) progression. Class I HDACs 1-3 are critical for activating AR-mediated transcription. Thus, targeting these HDACs is a promising strategy for treating PCa. Notably, along with significant adverse effects, several FDA-approved HDAC inhibitors broadly inhibiting different HDACs were ineffective for treating castration-resistant prostate cancer in clinical trials. Significantly, entinostat, an aminobenzamide analog specific to HDACs 1-3, extended overall survival for patients with breast cancer resistant to endocrine therapy. These observations suggest that HDACi selective to HDACs 1-3 may be effective for treating solid tumors including PCa. We have recently discovered the novel benzoylhydrazide class of HDAC inhibitors highly specific to HDACs 1-3. An optimized analog, SR-4370, exhibited low µM to nM potency against HDACs 1-3. SR-4370 markedly suppressed AR signaling, PCa cell proliferation in vitro, and prostate tumor growth in vivo. Gene expression profiling experiments revealed that SR-4370 downregulated AR, AR-Vs and AR target genes as well as the MYC oncogenic network. Chromatin accessibility assay using ATAC-seq showed that SR-4370 altered chromatin states in PCa cells. The chromatins with AR-binding sites became inaccessible on SR-4730 treatment, indicating that altered chromatin accessibility may contribute to the inhibition of AR signaling. Interestingly, SR-4370 sensitized C4-2 cells to enzalutamide. In PCa xenograft models, SR-4370 was effective to suppress tumor growth in vivo. Importantly, SR-4370 was well tolerated and did not cause observable adverse effects as judged by body weight and blood chemistry tests of treated mice. Our data suggest that SR-4370 may be a safe and clinically applicable treatment for advanced PCa refractory to current frontline treatments. (Supported by UFHealth Cancer Center, Florida Breast Cancer Foundation, James and Esther King Biomedical Research Program, and Bankhead-Coley Cancer Research Program, Florida Department of Health) Citation Format: Iqbal Mahmud, Guimei Tian, Jia Wang, Ryan Stowe, Zhiguang Huo, Yushan Zhang, Hamsa Thayek Purayil, Eric Helm, Theodore Drashansky, Dorina Avram, Yehia Daaka, William R. Roush, Daiqing Liao. SR-4370, a potent and selective inhibitor of class I HDACs, suppresses AR signaling and in vivo prostate tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4718.

Background: Advanced prostate cancer is treated by androgen deprivation, but despite initial responses most tumors inevitably recur. The recurrent disease is termed castration-resistant prostate cancer (CRPC) and is characterized by active androgen receptor (AR) signaling despite decreased androgen levels. AR activity in CRPC occurs through a variety of mechanisms including upregulation of AR coactivators, such as Vav3, and expression of constitutively active AR variants, which lack the ligand binding domain and are linked to poor prognosis. We previously demonstrated that PC cells are growth inhibited by depletion of either Vav3 or the clinically prevalent AR variant, AR-V7 (1). Gene expression profiling in CRPC cells revealed that arginine vasopressin receptor 1a (AVPR1a) was significantly reduced by depletion of either Vav3 or AR-V7. AVPR1a is a G protein-coupled receptor that has not been previously linked to PC. Analysis of publicly available datasets showed that the AVPR1a gene exhibits a significant increase in copy number in human CRPC specimens and AVPR1a mRNA is upregulated in aggressive PC (2-4). Methods: To explore the role of AVPR1a in PC cells, we selectively depleted AVPR1a in a panel of prostate cancer and prostate epithelial cell lines and examined cell survival and proliferation in culture and soft agar, as well as cell migration and invasion. Conversely, we overexpressed AVPR1a in an androgen-dependent PC cell line, LNCaP, and examined growth in vitro and in vivo in androgen-depleted conditions. We investigated AVPR1a antagonism in two distinct in vivo models of CRPC progression and growth using an effective and safe-in-humans AVPR1a antagonist, relcovaptan. Results: We showed that depletion of AVPR1a greatly inhibited the proliferation of multiple CRPC cell lines but had less to no effect on androgen-dependent PC cells or nontumorigenic prostate epithelial cells. Depletion of AVPR1a also decreased CRPC cell anchorage-independent growth. Conversely, overexpression of AVPR1a in androgen-dependent LNCaP cells conferred castration resistance in vitro and in vivo. Similar to depletion of AVPR1a, treatment of PC cells with the AVPR1a antagonist relcovaptan decreased CRPC anchorage-independent growth. AVPR1a depletion or antagonism was also effective at decreasing CRPC metastatic properties: migration and invasion. In a preclinical xenograft model of progression to castration resistance, we found that relcovaptan halted tumor growth and stabilized circulating levels of a clinical tumor progression marker, prostate specific antigen (PSA). We evaluated relcovaptan efficacy on end-stage metastatic CRPC growth. C4-2B CRPC cells were injected into the tibias of castrated mice to model tumor growth and bone remodeling. Relcovaptan significantly inhibited tumor growth and prevented cancer cell-induced bone remodeling. Conclusion: AVPR1a is a promising new therapeutic target against CRPC. We propose that AVPR1a antagonists may be repurposed for PC therapy. References: 1. Peacock SO, et al. Mol Endocrinol 2012;12:1967-79. 2. Chandran UR, et al. BMC Cancer 2007;7:64. 3. Tamura K, et al. BMC Cancer 2007;67:5117-25. 4. Beltran H, et al. Nat Med 2016;22;298-305. Citation Format: Ning Zhao, Stephanie Peacock, Chen Hao Lo, Laine Heidman, Fiorella Magani, Meghan Rice, Ann Greene, Yushan Zhang, Yehia Daaka, Conor Lynch, Kerry Burnstein. Preclinical evaluation of an arginine vasopressin receptor 1A (AVPR1A) antagonist in castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A063.

Class I HDACs 1-3 are critical for the AR-mediated transcriptional program. Thus, targeting class I HDACs is a promising strategy for treating prostate cancer. However, several FDA-approved HDAC inhibitors (HDACi) broadly inhibiting different HDACs have been shown to be ineffective and overtly toxic for treating castration-resistant prostate cancer in clinical trials. These observations suggest that HDACi selective for class I HDACs may be more effective but less toxic for treating prostate cancer than pan HDACi. Notably, we and others have shown that HDACi of different chemical classes individually exhibit nonoverlapping but limited effects on the acetylome in cancer cells. We hypothesize that a combination treatment strategy with class I HDAC-selective HDACi of different chemical classes is more effective to block AR signaling and prostate tumor growth than such HDACi individually. We have recently discovered the novel benzoylhydrazide chemo-type of class I HDAC-selective HDACi with a unique pharmacologic profile. Medicinal chemistry effort led to an optimized analog, SR-4370, with 6 nM potency to HDAC3. In support of our hypothesis, SR-4370 in combination with the clinical class I HDACi entinostat markedly suppressed AR signaling, prostate cancer cell proliferation in vitro, and tumor growth in vivo, and the tumor-suppressive effects by the HDACi combination were more pronounced than by SR-4370 and entinostat individually. Gene expression profiling experiments revealed that the SR-4370/entinostat combination profoundly downregulated AR, AR-V7, and AR target genes, while activating immune and cell death signaling pathways. In a C4-2 xenograft model using athymic nude mice, the SR-4370/entinostat combination was highly effective in suppressing tumor growth. Importantly, these HDACi alone or in combination did not cause observable adverse effects judged by body weight and blood chemistry tests of treated mice. Overall, our data suggest that the SR-4370/entinostat combination may be a translatable treatment for castration-resistant prostate cancer. Citation Format: Guimei Tian, Iqbal Mahmud, Ryan Stowe, Yushan Zhang, Hamsa Thayek Purayil, William Roush, Yehia Daaka, Daiqing Liao. A combination of two chemically distinct inhibitors of class I HDACs is highly effective to suppress AR signaling and in vivo growth of prostate cancer xenograft [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B004.

Renal Cell Carcinoma (RCC) is one of the most lethal urological cancers worldwide, with incidence and mortality rates increasing in the past two decades. The disease does not present early clinical symptoms and is commonly diagnosed at the metastatic stage, leaving the 5-year survival rate at ≈ 10-20%. For a wide array of neoplasms, many key molecular determinants involved in mediating the process of tumor cell growth, invasion, and colonization at a secondary site (i.e. metastasis) have been reported. However, few molecular predictors have been identified for RCC, rendering the mechanism(s) underlying RCC metastases poorly understood. This results in the lack of effective treatment for patients with advanced RCC. G protein-coupled receptors (GPCRs) and their effectors, such as the arrestin proteins, have been implicated in tumor growth, metastasis, and angiogenesis. Arrestin proteins are well known for their function in the desensitization and trafficking of GPCRs, but have also been implicated in unique signaling pathways to regulate fundamental cellular functions, including cell cycle progression, cell migration, and survival. Furthermore, arrestin involvement has been identified in a number of breast, colorectal, lung, and hematological malignancies. However, the role of arrestin proteins in RCC is yet to be determined. Our preliminary data show that β-arrestin 2 (βarr2) protein levels correlate with growth and metastatic potential in several RCC cell lines, including ACHN and SN12C. We hypothesize that βarr2 regulates RCC tumor progression, specifically through involvement in proliferation, invasion, and metastatic processes. To test our hypothesis, we used genetic-based loss of function approaches such as interfering RNA and CRISPR/Cas9. βarr2 knockdown results indicated a role in RCC malignancy as it significantly reduces the migration and invasion of RCC cell lines in vitro. βarr2 knockout (KO) impaired 3D spheroid formation of these cells and induced morphological changes compared to control cells as observed under confocal microscopy imaging. Moreover, epithelial marker E-cadherin expression levels were elevated while mesenchymal markers twist1, twist2 and vimentin levels were decreased in the βarr2 KO cells compared to control, indicating a mesenchymal to epithelial transition. In vivo data support our hypothesis that βarr2 plays a critical role in tumor growth and metastasis. Our data suggests a role for βarr2 in RCC malignancy and present a possible target in development of therapies for patients with advanced RCC. Citation Format: Jude Masannat, Yushan Zhang, Hamsa Purayil, Iqbal Mahmud, Yehia Daaka. β-arrestin 2 mediates tumor growth and metastasis in renal cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1982. doi:10.1158/1538-7445.AM2017-1982

The Cyclooxygenase-2 product PGE2 is suggested to be involved in angiogenesis, but its exact role and the underlying mechanisms remain unclear. In the present study, we show that PGE2 dose-dependently promotes the in vitro tube formation of human microvascular endothelial cells (HMVEC). PGE2 also increases vessel density in chicken chorioallantoic membrane, and vessel outgrowth of mouse aortic rings. All cognate PGE2 receptors, namely EP1, EP2, EP3 and EP4 can be detected in the HMVECs at both the gene and protein levels. Using EP-specific ligand agonists and antagonists our data demonstrate the EP4-dependent tube formation. Concordantly, knockdown of EP4, but not EP1, EP2 or EP3 with siRNA attenuates the PGE2-induced tube formation. Treatment with PGE2 increases cAMP accumulation and PKA activation, and knockdown of EP4 attenuates the PKA activation, suggesting a principally EP4-to-PKA signal. Remarkably, the treatment with selective PKA inhibitors H89 or PKI attenuates the PGE2-mediated tube formation. There are three PKA catalytic subunits and the knockdown of catalytic subunit gamma (PKACg) prevents the PGE2-regulated tube formation. To begin to identify the PKA substrate(s) involved in tube formation, a panel of SMARTpool siRNAs targeting 16 PKA substrates was tested. Results show that heat shock protein B6, endothelial nitric oxide synthase and small GTPase Rap1A exert pro-tube formation effects, whereas small GTPase RhoA and GSK3b may be involved in anti-tube formation. Our data suggest that PGE2 promotes angiogenesis through a EP4-cAMP-PKACgamma pathway. Targeting EP4 and PKACgamma may serve as an efficient strategy to control initiation and progression of diseases affected by angiogenesis, such as cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3488. doi:10.1158/1538-7445.AM2011-3488