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Stapled peptide inhibitors of RAB25 target context-specific phenotypes in cancer

ABSTRACT: The Ras-family small GTPase RAB25 is involved in numerous aspects of endosomal protein trafficking and cell polarity. Recent evidence has established a role for RAB25, as well as related RAB-family and effector proteins, in oncogenic signaling, highlighting the need for chemical probes targeting this class of proteins. Here we report the development of all-hydrocarbon stabilized peptides targeting RAB25 derived from the RAB-binding FIP-family of proteins. Relative to unmodified FIP peptides, optimized stapled peptides show markedly increased structural stability, binding affinity, cell permeability and inhibition of RAB25:FIP complex formation. RAB25 expression has been shown to promote both pro- and anti-oncogenic phenotypes in specific cellular contexts. Stapled peptide RFP14 treatment of breast and ovarian cancer cell lines, in which RAB25 is pro-oncogenic, inhibited migration and proliferation in a RAB25-dependent manner. In contrast, treatment of a triple-negative breast cancer cell line in which RAB25 is tumor suppressive augmented proliferation and migration. Gene expression (RNA Seq) profiling identified significantly altered transcripts in response to RAB25 expression in ovarian cancer cells, and treatment with the optimized stapled peptide RFP14 reversed this expression profile. These data validate first-in-class chemical probes targeting RAB-family proteins and support the role of RAB25 in regulating context-specific oncogenic phenotypes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE101528 | GEO | 2017/07/18

SECONDARY ACCESSION(S): PRJNA394735

REPOSITORIES: GEO

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Project description:Androgen receptor (AR) orchestrates an intricate transcriptional regulatory network that governs prostate cancer initiation, development and progression. To understand this network in detail, we generated genome-wide maps of AR occupancy by ChIP-seq in LNCaP cells. We found NKX3-1, an androgen-dependent homeobox protein well-characterized for its role in prostate development and differentiation, being recruited to AR binding sites (ARBS) in response to androgen signaling. We identified 6,359 NKX3-1 binding sites, most of which overlapped with AR. In addition to its novel collaborative transcriptional role at well-known prostate cancer model genes, our binding and knockdown studies further suggested that NKX3-1 potentially regulates AR in a feed-forward manner. Integrative analysis of Oncomine molecular concepts showed that these androgen-regulated AR and NKX3-1 associated genes are significantly overexpressed in prostate carcinoma as well as advanced and recurrent prostate tumors. From our transcriptomic profiling and Gene Ontology analysis, we observed that AR and NKX3-1 co-regulate genes involved in "protein trafficking" processes, which are mandatory events in the integration of oncogenic signaling pathways leading to prostate cancer development and progression. Interestingly, we found that AR and NKX3-1 co-regulate several members of the RAB GTPase family of secretory/trafficking proteins via the involvement of FoxA1 in a ternary complex and we believe that these AR/NKX3-1/FoxA1 co-regulated RAB genes could serve as expression signatures in prostate carcinogenesis. More specifically, through functional analyses, we showed that NKX3-1, together with AR and FoxA1, could promote prostate cancer cell survival through activation of RAB3B expression. Collectively, our study has provided important insights into the hierarchical transcriptional regulatory network established between AR and NKX3-1 and sought to elucidate the important genetic-molecular-phenotypic paradigm in androgen-dependent prostate cancer. Genome-wide binding analyses of AR, NKX3-1 and FoxA1 in LNCaP with or without DHT (5alpha-dihydrotestosterone) stimulation using ChIP-Seq.

Project description:Androgen receptor (AR) orchestrates an intricate transcriptional regulatory network that governs prostate cancer initiation, development and progression. To understand this network in detail, we generated genome-wide maps of AR occupancy by ChIP-seq in LNCaP cells. We found NKX3-1, an androgen-dependent homeobox protein well-characterized for its role in prostate development and differentiation, being recruited to AR binding sites (ARBS) in response to androgen signaling. We identified 6,359 NKX3-1 binding sites, most of which overlapped with AR. In addition to its novel collaborative transcriptional role at well-known prostate cancer model genes, our binding and knockdown studies further suggested that NKX3-1 potentially regulates AR in a feed-forward manner. Integrative analysis of Oncomine molecular concepts showed that these androgen-regulated AR and NKX3-1 associated genes are significantly overexpressed in prostate carcinoma as well as advanced and recurrent prostate tumors. From our transcriptomic profiling and Gene Ontology analysis, we observed that AR and NKX3-1 co-regulate genes involved in ‘protein trafficking’ processes, which are mandatory events in the integration of oncogenic signaling pathways leading to prostate cancer development and progression. Interestingly, we found that AR and NKX3-1 co-regulate several members of the RAB GTPase family of secretory/trafficking proteins via the involvement of FoxA1 in a ternary complex and we believe that these AR/NKX3-1/FoxA1 co-regulated RAB genes could serve as expression signatures in prostate carcinogenesis. More specifically, through functional analyses, we showed that NKX3-1, together with AR and FoxA1, could promote prostate cancer cell survival through activation of RAB3B expression. Collectively, our study has provided important insights into the hierarchical transcriptional regulatory network established between AR and NKX3-1 and sought to elucidate the important genetic-molecular-phenotypic paradigm in androgen-dependent prostate cancer. Gene expression profiling of LNCaP in response to ETOH (vehicle) or DHT (5α-dihydrotestosterone) stimulation across different treatment time-points using microarray.

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Stapled peptide inhibitors of RAB25 target context-specific phenotypes in cancer

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