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Supplementary Figure 3

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PDF file - 76K, Supplemental Table 1. Clinicopathologic characteristics of prostate cancer patients. Supplemental Table 2. Complimentary sequences in 3'UTRs of genes for miR-34b and sequences of primers and probes.

Legends to Supplementary Data

Contains Supplementary Table 1, and Supplementary Figure 1 and 2

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Supplementary Table 2

PDF file - 325K, Supplemental Figure 1: Downregulation of miR-34b expression, its potential diagnostic and prognostic significance and methylation status in PCa. A) Box plot representation of mir-34b expression in a cohort of unmatched tissue samples. B) Box plot representation of ISH results of tissue samples. (Ave- Average; T/N- Tumor/Normal). C) Chi-square test showing correlation of clinicopathological characteristics with miR-34b expression. D) CpG islands within the 1.0 kb region upstream of the miR-34b gene. Arrow indicates the start of the miR-34b gene. Locations for primers and probes are also shown on the miR-34b gene. F- Forward, R-Reverse, M/U Methylation and Unmethylation probes, CF1, CF2, CR1, CR2- ChiP Forward1-2, ChIP Reverse 1-2. E) Correlation of miR-34b expression with average percent methylation indicating cases with low miR-34b expression have higher methylation and vice versa.

Purpose: Previous studies have indicated an important role for pleckstrin homology domain-interacting protein (PHIP) as a marker and mediator of melanoma metastasis. Here we aimed to confirm the role of PHIP copy number in successive stages of melanoma progression.Experimental Design: PHIP copy number was examined using FISH in three independent cohorts by recording the percentage of cells harboring ≥3 copies of PHIP. The impact of PHIP copy number on survival was assessed using Cox regression analysis. The enrichment of PHIP was assessed in various molecular melanoma subtypes. PHIP expression was analyzed in The Cancer Genome Atlas (TCGA) melanoma cohort.Results: Elevated PHIP copy number was significantly predictive of reduced distant metastasis-free survival (DMFS) and disease-specific survival (DSS), and increased prevalence of ulceration in primary melanoma (cohort No. 1). By multivariate analysis, PHIP FISH scores were independently predictive of DMFS and DSS. PHIP copy number was enriched in metastatic melanomas harboring mutant NRAS or expressing PTEN protein (cohort No. 2). PHIP copy number was significantly elevated in metastatic melanomas when compared with matched primary tumors from the same patient (cohort No. 3). Several of these associations were replicated using TCGA cohort analysis.Conclusions: These results underscore the important role of PHIP copy-number elevation in melanoma progression, and identify molecular subtypes of melanoma in which PHIP is enriched. Finally, as elevated PHIP copy number appears to be selected for during the progression of primary to metastatic melanoma, these results confirm PHIP as a promising therapeutic target for melanoma. Clin Cancer Res; 24(17); 4119–25. ©2018 AACR.

Melanoma occurs as a consequence of inherited susceptibility to the disease and exposure to UV radiation (UVR) and is characterized by uncontrolled cellular proliferation and a high mutational load. The precise mechanisms by which UVR contributes to the development of melanoma remain poorly understood. Here we show that activation of nuclear receptor coactivator 3 (NCOA3) promotes melanomagenesis through regulation of UVR sensitivity, cell-cycle progression, and circumvention of the DNA damage response (DDR). Downregulation of NCOA3 expression, either by genetic silencing or small-molecule inhibition, significantly suppressed melanoma proliferation in melanoma cell lines and patient-derived xenografts. NCOA3 silencing suppressed expression of xeroderma pigmentosum C and increased melanoma cell sensitivity to UVR. Suppression of NCOA3 expression led to activation of DDR effectors and reduced expression of cyclin B1, resulting in G2–M arrest and mitotic catastrophe. A SNP in NCOA3 (T960T) reduced NCOA3 protein expression and was associated with decreased melanoma risk, given a significantly lower prevalence in a familial melanoma cohort than in a control cohort without cancer. Overexpression of wild-type NCOA3 promoted melanocyte survival following UVR and was accompanied by increased levels of UVR-induced DNA damage, both of which were attenuated by overexpression of NCOA3 (T960T). These results describe NCOA3-regulated pathways by which melanoma can develop, with germline NCOA3 polymorphisms enabling enhanced melanocyte survival in the setting of UVR exposure, despite an increased mutational burden. They also identify NCOA3 as a novel therapeutic target for melanoma.Significance:This study explores NCOA3 as a regulator of the DDR and a therapeutic target in melanoma, where activation of NCOA3 contributes to melanoma development following exposure to ultraviolet light.

PDF file - 141K, miR-23b directly targets Akt in prostate cancer

Supplementary Figure Legend from Regulation of Minichromosome Maintenance Gene Family by MicroRNA-1296 and Genistein in Prostate Cancer

PDF file - 65K, Clinicopathological characteristics of patients and their correlation with miR-23b

PDF file - 967K, Depletion of Src kinase by an additional siRNA sequence

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Supplementary Figure 4 from MicroRNA-205 Inhibits Src-Mediated Oncogenic Pathways in Renal Cancer

Supplementary Figure Legends 1-4 from MicroRNA-205 Inhibits Src-Mediated Oncogenic Pathways in Renal Cancer

Supplementary Figure 1 from Regulation of Minichromosome Maintenance Gene Family by MicroRNA-1296 and Genistein in Prostate Cancer

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Supplementary Figure 3 from MicroRNA-205 Inhibits Src-Mediated Oncogenic Pathways in Renal Cancer

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Supplementary Figure 2 from MicroRNA-205 Inhibits Src-Mediated Oncogenic Pathways in Renal Cancer

Supplementary Figure 1 from MicroRNA-205 Inhibits Src-Mediated Oncogenic Pathways in Renal Cancer

Supplementary Table 1 from MicroRNA-205 Inhibits Src-Mediated Oncogenic Pathways in Renal Cancer

We describe our institutional experience of developing a liquid biopsy approach using circulating tumor DNA (ctDNA) analysis for personalized medicine in cancer patients, focusing on the hurdles encountered during the multistep process in order to benefit other investigators wishing to set up this type of study in their institution. Blood samples were collected at the time of cancer surgery from 209 patients with one of nine different cancer types. Extracted tumor DNA and circulating cell-free DNA were sequenced using cancer-specific panels and the Illumina MiSeq machine. Almost half of the pairs investigated were uninformative, mostly because there was no trackable pathogenic mutation detected in the original tumor. The pairs with interpretable data corresponded to 107 patients. Analysis of 48 gene sequences common to both panels was performed and revealed that about 40% of these pairs contained at least one driver mutation detected in the DNA extracted from plasma. Here, we describe the choice of our overall approach, the selection of the cancer panels, and the difficulties encountered during the multistep process, including the use of several tumor types and in the data analysis. We also describe some case reports using longitudinal samples, illustrating the potential advantages and rewards in performing ctDNA sequencing to monitor tumor burden or guide treatment for cancer patients.

Background and aims Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors. Approach and results Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl4 treatment (JnkΔhepa + DEN + CCl4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in JnkΔhepa + DEN + CCl4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status. Conclusions Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.

Therapy of BRAF -mutant melanoma with selective inhibitors of BRAF (BRAFi) and MEK (MEKi) represents a major clinical advance but acquired resistance to therapy has emerged as a key obstacle. To date, no clinical approaches successfully resensitize to BRAF/MEK inhibition. Here, we develop a therapeutic strategy for melanoma using bromosporine, a bromodomain inhibitor. Bromosporine (bromo) monotherapy produced significant anti-tumor effects against established melanoma cell lines and patient-derived xenografts (PDXs). Combinatorial therapy involving bromosporine and cobimetinib (bromo/cobi) showed synergistic anti-tumor effects in multiple BRAFi-resistant PDX models. The bromo/cobi combination was superior in vivo to standard BRAFi/MEKi therapy in the treatment-naive BRAF -mutant setting and to MEKi alone in the setting of immunotherapy-resistant NRAS - and NF1 -mutant melanoma. RNA sequencing of xenografts treated with bromo/cobi revealed profound down-regulation of genes critical to cell division and mitotic progression. Bromo/cobi treatment resulted in marked DNA damage and cell-cycle arrest, resulting in induction of apoptosis. These studies introduce bromodomain inhibition, alone or combined with agents targeting the mitogen activated protein kinase pathway, as a rational therapeutic approach for melanoma refractory to standard targeted or immunotherapeutic approaches.

The invasive behavior of glioblastoma is essential to its aggressive potential. Here, we show that pleckstrin homology domain interacting protein (PHIP), acting through effects on the force transduction layer of the focal adhesion complex, drives glioblastoma motility and invasion. Immunofluorescence analysis localized PHIP to the leading edge of glioblastoma cells, together with several focal adhesion proteins: vinculin (VCL), talin 1 (TLN1), integrin beta 1 (ITGB1), as well as phosphorylated forms of paxillin (pPXN) and focal adhesion kinase (pFAK). Confocal microscopy specifically localized PHIP to the force transduction layer, together with TLN1 and VCL. Immunoprecipitation revealed a physical interaction between PHIP and VCL. Targeted suppression of PHIP resulted in significant down-regulation of these focal adhesion proteins, along with zyxin (ZYX), and produced profoundly disorganized stress fibers. Live-cell imaging of glioblastoma cells overexpressing a ZYX-GFP construct demonstrated a role for PHIP in regulating focal adhesion dynamics. PHIP silencing significantly suppressed the migratory and invasive capacity of glioblastoma cells, partially restored following TLN1 or ZYX cDNA overexpression. PHIP knockdown produced substantial suppression of tumor growth upon intracranial implantation, as well as significantly reduced microvessel density and secreted VEGF levels. PHIP copy number was elevated in the classical glioblastoma subtype and correlated with elevated EGFR levels. These results demonstrate PHIP’s role in regulating the actin cytoskeleton, focal adhesion dynamics, and tumor cell motility, and identify PHIP as a key driver of glioblastoma migration and invasion.

Background Patient-derived xenograft (PDX) mouse tumour models can predict response to therapy in patients. Predictions made from PDX cultures (PDXC) would allow for more rapid and comprehensive evaluation of potential treatment options for patients, including drug combinations. Methods We developed a PDX library of BRAF-mutant metastatic melanoma, and a high-throughput drug-screening (HTDS) platform utilising clinically relevant drug exposures. We then evaluated 34 antitumor agents across eight melanoma PDXCs, compared drug response to BRAF and MEK inhibitors alone or in combination with PDXC and the corresponding PDX, and investigated novel drug combinations targeting BRAF inhibitor-resistant melanoma. Results The concordance of cancer-driving mutations across patient, matched PDX and subsequent PDX generations increases as variant allele frequency (VAF) increases. There was a high correlation in the magnitude of response to BRAF and MEK inhibitors between PDXCs and corresponding PDXs. PDXCs and corresponding PDXs from metastatic melanoma patients that progressed on standard-of-care therapy demonstrated similar resistance patterns to BRAF and MEK inhibitor therapy. Importantly, HTDS identified novel drug combinations to target BRAF-resistant melanoma. Conclusions The biological consistency observed between PDXCs and PDXs suggests that PDXCs may allow for a rapid and comprehensive identification of treatments for aggressive cancers, including combination therapies.

Simple Summary Cholangiocarcinoma (CCA) is a rare and highly aggressive tumor with limited therapeutic options, thus underscoring the need to develop novel therapeutic approaches. We analyzed a publicly available CCA patient database to identify mutations in DNA damage response (DDR) genes. Mutations in DDR genes were prevalent, thus rendering these tumors potentially susceptible to poly-ADP-ribose polymerase (PARP) inhibition. PARP genes are critical to DNA repair and genomic stability. The role of PARP inhibitors in CCA was investigated by employing a series of in vitro functional assays and in vivo patient-derived xenograft models. This study highlights the therapeutic potential of PARP inhibitors alone or in combination with the chemotherapeutic agent gemcitabine for the treatment of CCA. Abstract Cholangiocarcinoma (CCA) is the second most common hepatobiliary cancer, an aggressive malignancy with limited therapeutic options. PARP (poly (ADP-ribose) polymerase) 1 and 2 are important for deoxyribonucleotide acid (DNA) repair and maintenance of genomic stability. PARP inhibitors (PARPi) such as niraparib have been approved for different malignancies with genomic alteration in germline BRCA and DNA damage response (DDR) pathway genes. Genomic alterations were analyzed in DDR genes in CCA samples employing The Cancer Genome Atlas (TCGA) database. Mutations were observed in various DDR genes, and 35.8% cases had alterations in at least one of three genes (ARID1A, BAP1 and ATM), suggesting their susceptibility to PARPi. Niraparib treatment suppressed cancer cell viability and survival, and also caused G2/M cell cycle arrest in patient-derived xenograft cells lines (PDXC) and established CCA cells harboring DDR gene mutations. PARPi treatment also induced apoptosis and caspase3/7 activity in PDXC and CCA cell lines, and substantially reduced expression of BCL2, BCL-XL and MCL1 proteins. Niraparib caused a significant increase in oxidative stress, and induced activation of DNA damage markers, phosphorylation of CHK2 and replication fork stalling. Importantly, niraparib, in combination with gemcitabine, produced sustained and robust inhibition of tumor growth in vivo in a patient-derived xenograft (PDX) model more effectively than either treatment alone. Furthermore, tissue samples from mice treated with niraparib and gemcitabine display significantly lower expression levels of pHH3 and Ki-67, which are a mitotic and proliferative marker, respectively. Taken together, our results indicate niraparib as a novel therapeutic agent alone or in combination with gemcitabine for CCA.

Melanoma occurs as a consequence of inherited susceptibility to the disease and exposure to UV radiation (UVR) and is characterized by uncontrolled cellular proliferation and a high mutational load. The precise mechanisms by which UVR contributes to the development of melanoma remain poorly understood. Here we show that activation of nuclear receptor coactivator 3 (NCOA3) promotes melanomagenesis through regulation of UVR sensitivity, cell-cycle progression, and circumvention of the DNA damage response (DDR). Downregulation of NCOA3 expression, either by genetic silencing or small-molecule inhibition, significantly suppressed melanoma proliferation in melanoma cell lines and patient-derived xenografts. NCOA3 silencing suppressed expression of xeroderma pigmentosum C and increased melanoma cell sensitivity to UVR. Suppression of NCOA3 expression led to activation of DDR effectors and reduced expression of cyclin B1, resulting in G2–M arrest and mitotic catastrophe. A SNP in NCOA3 (T960T) reduced NCOA3 protein expression and was associated with decreased melanoma risk, given a significantly lower prevalence in a familial melanoma cohort than in a control cohort without cancer. Overexpression of wild-type NCOA3 promoted melanocyte survival following UVR and was accompanied by increased levels of UVR-induced DNA damage, both of which were attenuated by overexpression of NCOA3 (T960T). These results describe NCOA3-regulated pathways by which melanoma can develop, with germline NCOA3 polymorphisms enabling enhanced melanocyte survival in the setting of UVR exposure, despite an increased mutational burden. They also identify NCOA3 as a novel therapeutic target for melanoma. Significance: This study explores NCOA3 as a regulator of the DDR and a therapeutic target in melanoma, where activation of NCOA3 contributes to melanoma development following exposure to ultraviolet light.

Cholangiocarcinoma (CCA) is a rare, highly invasive malignancy, and its incidence is increasing globally. MicroRNAs (miRNAs) mediate a wide array of cellular and biological processes and are dysregulated in various tumors. The functional and biological roles of miRNAs in CCA have not been fully elucidated. In this study, we show that miR-876 expression levels and copy number are significantly attenuated in the TCGA cohort of CCA tissue samples. TCGA expression data was consistent with the observed substantial decrease in miR-876 expression in patient samples and CCA cell lines. In-silico algorithm databases revealed BCL-XL as a potential target of miR-876. We observed miR-876 expression to be downregulated, whereas, BCL-XL upregulated in CCA cell lines. BCL-XL was identified as a direct functional target of miR-876 in CCA. miR-876-mediated reduction of BCL-XL regulated cell survival, induced apoptosis and caspase 3/7 expression in CCA. BCL-XL overexpression reversed the miR-876 mediated effect on CCA cell growth and apoptosis. Stable overexpression of miR-876 produced potent tumor suppressor activity and in vivo tumor cell growth reduction. Overexpression of miR-876 in a patient-derived xenograft (PDX) cell line significantly suppressed BCL-XL expression and spheroid formation with a concomitant induction of caspase 3/7 activity and apoptosis. This study demonstrates a novel tumor suppressor role for miR-876 in CCA, identifies BCL-XL as an actionable target, and suggests a potential therapeutic role for miR-876 in CCA.

Cholangiocarcinoma (CCA) is a highly invasive cancer, diagnosed at an advanced stage, and refractory to surgical intervention and chemotherapy. Cyclin-dependent kinases (CDKs) regulate cell cycle progression and transcriptional processes, and are considered potential therapeutic targets for cancer. Dinaciclib is a small molecule multi-CDK inhibitor targeting CDK 2/5/9. In this study, the therapeutic efficacy of dinaciclib was assessed using patient-derived xenograft cells (PDXC) and CCA cell lines. Treatment with dinaciclib significantly suppressed cell proliferation, induced caspase 3/7 levels and apoptotic activity in PDXC and CCA cell lines. Dinaciclib suppressed expression of its molecular targets CDK2/5/9, and anti-apoptotic BCL-XL and BCL2 proteins. Despite the presence of cyclin D1 amplification in the PDXC line, palbociclib treatment had no effect on cell proliferation, cell cycle or apoptosis in the PDXC as well as other CCA cell lines. Importantly, dinaciclib, in combination with gemcitabine, produced a robust and sustained inhibition of tumor progression in vivo in a PDX mouse model, greater than either of the treatments alone. Expression levels of two proliferative markers, phospho-histone H3 and Ki-67, were substantially suppressed in samples treated with the combination regimen. Our results identify dinaciclib as a novel and potent therapeutic agent alone or in combination with gemcitabine for the treatment of CCA.

Homologous recombination DNA damage repair (HR-DDR) deficient patients with various solid tumors have been treated with PARP inhibitors. However, the clinical characteristics of patients with melanoma who have HR-DDR gene mutations and the consequences of PARP inhibition are poorly understood. We compared the commercially available next-generation sequencing data from 84 patients with melanomas from our institution with a dataset of 1,986 patients as well as 1,088 patients profiled in cBioportal. In total, 21.4% of patients had ≥1 functional HR-DDR mutation, most commonly involving BRCA1, ARID1A, ATM, ATR, and FANCA. Concurrent NF1, BRAF, and NRAS mutations were found in 39%, 39%, and 22% of cases, respectively. HR-DDR gene mutation was associated with high tumor mutational burden and clinical response to checkpoint blockade. A higher prevalence of HR-DDR mutations was observed in the datasets from Foundation Medicine (Cambridge, CA) and those from the Cancer Genome Atlas. Treatment of HR-DDR‒mutated patient-derived xenograft models of melanoma with PARP inhibitor produced significant antitumor activity in vivo and was associated with increased apoptotic activity. RNA sequencing analysis of PARP inhibitor–treated tumors indicated alterations in the pathways involving extracellular matrix remodeling, cell adhesion, and cell-cycle progression. Melanomas with HR-DDR mutations represent a unique subset, which is more likely to benefit from checkpoint blockade and may be targeted with PARP inhibitor.

Prostate carcinogenesis involves alterations in several signaling pathways, the most prominent being the PI3K/AKT pathway. This pathway is constitutively active and drives prostate cancer (PCa) progression to advanced metastatic disease. PTEN, a critical tumor and metastasis suppressor gene negatively regulates cell survival, proliferation, migration and angiogenesis via the PI3K/Akt pathway. PTEN is mutated, downregulated/dysfunctional in many cancers and its dysregulation correlates with poor prognosis in PCa. Here, we demonstrate that microRNA-4534 (miR-4534) is overexpressed in PCa and show that miR-4534 is hypermethylated in normal tissues and cell lines compared to PCa tissues/cells. miR-4534 exerts its oncogenic effects partly by downregulating the tumor suppressor PTEN gene. Knockdown of miR-4534 impaired cell proliferation, migration/invasion and induced G0/G1 cell cycle arrest and apoptosis in PCa. Suppression of miR-4534 and its effects on tumor growth was confirmed in a xenograft mouse model. We performed parallel experiments in non-cancer RWPE1 cells by overexpessing miR-4534 followed by functional assays. Overexpression of miR-4534 induced pro-cancerous characteristics in this non-cancer cell line. Statistical analyses revealed that miR-4534 has potential to independently distinguish malignant from normal tissues and positively correlated with poor overall and PSA recurrence free survival. Taken together, our results show that depletion of miR-4534 in PCa induces a tumor suppressor phenotype partly through induction of PTEN. These results have important implications for identifying and defining the role of new PTEN regulators such as microRNAs in prostate tumorigenesis. Understanding aberrantly overexpressed miR-4534 and its downregulation of PTEN will provide mechanistic insight and therapeutic targets for PCa therapy.

The identification and targeting of key molecular drivers of melanoma and breast and lung cancer have substantially improved their therapy. However, subtypes of each of these three common, lethal solid tumors lack identified molecular drivers, and are thus not amenable to targeted therapies. Here we show that pleckstrin homology domain-interacting protein (PHIP) promotes the progression of these "driver-negative" tumors. Suppression of PHIP expression significantly inhibited both tumor cell proliferation and invasion, coordinately suppressing phosphorylated AKT, cyclin D1, and talin1 expression in all three tumor types. Furthermore, PHIP's targetable bromodomain is functional, as it specifically binds the histone modification H4K91ac. Analysis of TCGA profiling efforts revealed PHIP overexpression in triple-negative and basal-like breast cancer, as well as in the bronchioid subtype of nonsmall cell lung cancer. These results identify a role for PHIP in the progression of melanoma and breast and lung cancer subtypes lacking identified targeted therapies. The use of selective, anti-PHIP bromodomain inhibitors may thus yield a broad-based, molecularly targeted therapy against currently nontargetable tumors.

Purpose: Previous studies have indicated an important role for pleckstrin homology domain-interacting protein (PHIP) as a marker and mediator of melanoma metastasis. Here we aimed to confirm the role of PHIP copy number in successive stages of melanoma progression. Experimental Design: PHIP copy number was examined using FISH in three independent cohorts by recording the percentage of cells harboring ≥3 copies of PHIP. The impact of PHIP copy number on survival was assessed using Cox regression analysis. The enrichment of PHIP was assessed in various molecular melanoma subtypes. PHIP expression was analyzed in The Cancer Genome Atlas (TCGA) melanoma cohort. Results: Elevated PHIP copy number was significantly predictive of reduced distant metastasis-free survival (DMFS) and disease-specific survival (DSS), and increased prevalence of ulceration in primary melanoma (cohort No. 1). By multivariate analysis, PHIP FISH scores were independently predictive of DMFS and DSS. PHIP copy number was enriched in metastatic melanomas harboring mutant NRAS or expressing PTEN protein (cohort No. 2). PHIP copy number was significantly elevated in metastatic melanomas when compared with matched primary tumors from the same patient (cohort No. 3). Several of these associations were replicated using TCGA cohort analysis. Conclusions: These results underscore the important role of PHIP copy-number elevation in melanoma progression, and identify molecular subtypes of melanoma in which PHIP is enriched. Finally, as elevated PHIP copy number appears to be selected for during the progression of primary to metastatic melanoma, these results confirm PHIP as a promising therapeutic target for melanoma. Clin Cancer Res; 24(17); 4119–25. ©2018 AACR.

MicroRNAs (miRNAs) play a key role in cancer progression by coordinately repressing target genes involved in cell proliferation, migration, and invasion. miRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report that expression of miR-1280 is significantly suppressed in human melanoma specimens when compared with nevi, and in human melanoma cell lines when compared with cultured normal human melanocytes. The proto-oncogene Src was identified as a target of miR-1280 action. Levels of Src expression were significantly higher in melanoma samples and cell lines than in nevi and normal melanocytes. miR-1280 overexpression significantly suppressed the luciferase activity of reporter plasmids containing the full-length 3′ untranslated region of Src. miR-1280-mediated suppression of Src led to substantial decreases in melanoma cell proliferation, cell cycle progression, invasion, as well as induced melanoma cell apoptosis. The effects of miR-1280 overexpression on melanoma cell proliferation and growth were reversed by Src overexpression. Intratumoral delivery of miR-1280 significantly suppressed melanoma cell growth in vivo. Our results demonstrate a novel role for miR-1280 as a tumor suppressor in melanoma, identify the Src signaling pathway as a target of miR-1280 action, and suggest a potential therapeutic role for miR-1280 in melanoma. OA embargo

miRNAs are implicated in regulating cancer progression and metastasis. Here, we show that miR-720 is positively associated with renal cell carcinoma (RCC). Elevated levels of miR-720 were observed in a panel of RCC cell lines and clinical tissues compared with nonmalignant cell line and normal samples. Loss of miR-720 function inhibited proliferation, migration, and invasion and induced apoptosis in RCC cell lines in vitro and repressed tumor growth in xenograft mouse models. Conversely, gain of miR-720 function in nonmalignant HK-2 cells induced procancerous characteristics. Silencing of miR-720 caused a marked induction in the levels of endogenous αE-catenin and E-cadherin protein levels in anti720 transfected cells compared with control, whereas miR-720 overexpression in RCC cell lines reduced activity of a luciferase reporter gene fused to the wild-type αE-catenin or E-cadherin 3′UTR compared with nonspecific 3′UTR control, indicating that αE-catenin–E-cadherin complex is a direct and functional target of miR-720 in RCC. We also observed attenuation of β-catenin, CD44, and Akt expression in RCC cells transfected with miR-720 inhibitor compared with control. Furthermore, miR-720 exhibited clinical significance in RCC. Expression of miR-720 significantly distinguished malignant from normal samples. Elevated miR-720 levels positively correlated with higher Fuhrman grade, pathologic stage, and poor overall survival of RCC patients. These findings uncover a new regulatory network in RCC involving metastasis-promoting miR-720 that directly targets expression of key metastasis-suppressing proteins E-cadherin and αE-catenin complex. These results suggest that therapeutic regulation of miR-720 may provide an opportunity to regulate EMT and metastasis in RCC. Mol Cancer Ther; 16(12); 2840–8. ©2017 AACR.

Summary Melanoma is one of the most aggressive and deadly skin cancers, and, in its advanced stages, accounts for > 80% mortality. The incidence of melanoma is increasing worldwide; however, beyond surgical removal of the tumour, there is currently no curative therapy available, especially for its advanced stages. This may, in part, be owing to incomplete understanding of the molecular mechanisms that regulate the initiation and/or progression of melanoma to metastasis. The molecular mechanisms leading to the development and progression of melanoma are the focus of intense investigation, and many genetic/epigenetic alterations affecting melanoma progression and development have been identified. microRNAs (miRNAs) are emerging as important causal modulators in the development and progression of melanoma. The understanding of miRNA-mediated regulation of tumours has grown immensely over the last few years, as it has been understood to regulate most biological processes. Here, we review the currently available data on miRNAs associated with melanoma, highlighting those deregulated miRNAs that target important genes and pathways involved in the progression of melanocytes to primary and metastatic melanoma. We also review their potential clinical utility as biomarkers and potential use in targeted therapy.

Ulceration is an important prognostic factor in melanoma whose biologic basis is poorly understood. Here we assessed the prognostic impact of pleckstrin homology domain-interacting protein (PHIP) copy number and its relationship to ulceration. PHIP copy number was determined using fluorescence in situ hybridization (FISH) in a tissue microarray cohort of 238 melanomas. Elevated PHIP copy number was associated with significantly reduced distant metastasis-free survival (DMFS; P=0.01) and disease-specific survival (DSS; P=0.009) by Kaplan-Meier analyses. PHIP FISH scores were independently predictive of DMFS (P=0.03) and DSS (P=0.03). Increased PHIP copy number was an independent predictor of ulceration status (P=0.04). The combined impact of increased PHIP copy number and tumor vascularity on ulceration status was highly significant (P

Cadmium (Cd) has been implicated in cancer development and classified as a type I carcinogen by the International Agency for Cancer Research. The etiology of prostate cancer development is associated with multitude of causative risk factors including exposure to cadmium. However, the molecular mechanisms associated with Cd-induced prostate cancer remain elusive. This study provides evidence that PI3K/Akt signaling is a major molecular pathway involved in Cd induced malignant transformation of normal prostate epithelial cells. Functionally, Cd exposure induced aggressive behavior as indicated by increased proliferation, migration and invasion in Cd-RWPE1 cells compared to parental RWPE1. PI3K/Akt pathway is constitutively activated in prostate cancer driving the most aggressive forms of cancer and metastasis. Consistent with these findings, the RT2 PCR array analysis of 84 genes involved in the PI3K/Akt pathway revealed induction of gene expression in catalytic units (P110α, Akt, mTOR, NFKB1, RAF etc.) with a concomitant reduction in expression of regulatory units (PIK3R1, PIK3R2, PTEN etc.) of the PI3K/Akt pathway in Cd-RWPE1 cells compared to parental RWPE1. This was confirmed by individual quantitative real-time PCR analysis using TaqMan gene expression assay probes. Effect of Cd on the translation of the PI3K/Akt pathway genes was examined by immunoblot assays. Gene Set Enrichment Analysis (GSEA) for differentially expressed genes in Cd-RWPE1 showed 5 overlapping pathways that were enriched in Cd-treated cells (Cd-RWPE1) and negatively correlated with parental RWPE1. The overlapping pathways include KEGG Apoptosis pathway (ES=0.56, NES=1), KEGG ERBB pathway (ES=0.25, NES=1), KEGG MAPK pathway (ES=0.48, NES=1), KEGG Pathways in cancer (ES=0.33, NES=1) and KEGG Prostate Cancer pathway (ES=0.35, NES=1). Interestingly, all these pathways are implicated in prostate cancer progression and metastasis. We randomly selected up- and down-regulated genes from the PI3K/Akt pathway in PCR array and performed profile analysis in a prostate adenocarcinoma data set (n=534) from the TCGA/GDC data base. We observed upregulation of the oncogenes along with downregulation of tumor suppressors in prostate cancer compared to normal prostate samples. Taken together, these data reveal that Cd exposure induced aggressive malignant characteristics in normal prostate epithelial cells via modulation of the PI3K/Akt signaling pathway. Understanding the molecular mechanisms involved in the malignant transformation of normal prostate epithelial cells may help develop optimal therapeutic strategies for advanced prostate cancer. Citation Format: Priyanka Kulkarni, Pritha Dasgupta, Nadeem S. Bhat, Yutaka Hashimoto, Sharanjot Saini, Altaf A. Dar, Varahram Shahryari, Marisa Shiina, Soichiro Yamamura, Yuichiro Tanaka, Rajvir Dahiya, Shahana Majid. Involvement of PI3K/Akt pathway in cadmium triggered aggressive prostate cancer [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 5040.

Cadmium (Cd) is a chemical pollutant of the natural and occupational environment and is reported to be associated with human carcinogenesis. The molecular mechanisms associated with Cd-induced prostate cancer remain elusive. This study provides evidence that Erk/MAPK signaling is a carcinogenic molecular fingerprint for Cd induced prostate cancer. Cd exposed RWPE1 (Cd-RWPE1) cells robustly formed tumors in nude mice. Functionally, chronic Cd exposure of RWPE1 cells promoted cell survival, proliferation and colony formation with inhibition of apoptosis. RT2 PCR array analysis of 84 genes involved in the Erk/MAPK pathway revealed induction of gene expression in Cd-RWPE1 cells compared to RWPE1. This was confirmed by individual TaqMan gene expression analysis. Gene Set Enrichment Analysis (GSEA) for differentially expressed genes in Cd-RWPE1 showed an enrichment of the Erk/MAPK pathway along with other pathways such as KEGG-ERBB, KEGG-Cell Cycle, KEGG-VEGF, KEGG-Pathways in cancer and KEGG-prostate cancer pathway. We randomly selected upregulated genes from the Erk/MAPK pathway and performed profile analysis in a prostate adenocarcinoma data set (n=534) from the TCGA/GDC data base. We observed upregulation of these genes in prostate cancer compared to normal prostate samples. Taken together, these data reveal that Erk/MAPK signaling is a major pathway involved in Cd-induced malignant transformation of normal prostate epithelial cells. Understanding the dominant oncogenic pathways involved in the malignant transformation of normal prostate epithelial cells may help develop optimal therapeutic strategies for prostate cancer. Citation Format: Pritha Dasgupta, Priyanka Kulkarni, Nadeem S. Bhat, Ravi Gupta, Yutaka Hashimoto, Sharanjot Saini, Altaf A. Dar, Varahram Shahryari, Soichiro Yamamura, Yuichiro Tanaka, Marisa Shiina, Rajvir Dahiya, Shahana Majid. Cadmium induced malignant transformation involves activation of the Erk/MAPK pathway [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 4659.

Bromodomain PHD finger transcription factor (BPTF) plays an important role in chromatin remodeling, but its functional role in tumor progression is incompletely understood. Here we reveal new pro-oncogenic roles for BPTF in promoting tumor cell proliferation and resistance to targeted therapies. shRNA-mediated BPTF silencing suppressed the proliferative capacity (by 65.5%) and metastatic potential (by 66.4%) of melanoma cells. Elevated BPTF mean copy number greater than 3 was observed in 28 of 77 (36.4%) melanomas. BPTF overexpression predicted poor survival in a cohort of 311 melanoma patients (distant metastasis-free survival, p=0.03, and disease-specific survival p=0.008), and promoted resistance to BRAF inhibitors in melanoma cell lines. Metastatic melanoma tumors progressing on BRAF inhibitors contained low BPTF-expressing, apoptotic tumor cell sub-clones, indicating the continued presence of drug-responsive sub-clones within tumors demonstrating overall resistance to anti-BRAF agents. These studies demonstrate multiple pro-tumorigenic functions for BPTF, and identify it as a novel target for anti-cancer therapy. They also suggest the combination of BPTF targeting with BRAF inhibitors as a novel therapeutic strategy for melanomas with mutant BRAF. Citation Format: Altaf A. Dar, Shahana Majid, David de Semir, Vladimir Bezrookove, Mehdi Nosrati, Mohammed Kashani-Sabet. BPTF role in melanoma progression and BRAF resistance therapy [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 2607.