The landscape of alternative polyadenylation during EMT and its regulation by the RNA-binding protein Quaking.

Epithelial-mesenchymal transition (EMT) plays important roles in tumour progression and is orchestrated by dynamic changes in gene expression. While it is well established that post-transcriptional regulation plays a significant role in EMT, the extent of alternative polyadenylation (APA) during EMT has not yet been explored. Using 3' end anchored RNA sequencing, we mapped the alternative polyadenylation (APA) landscape following Transforming Growth Factor (TGF)-ß-mediated induction of EMT in human mammary epithelial cells and found APA generally causes 3'UTR lengthening during this cell state transition. Investigation of potential mediators of APA indicated the RNA-binding protein Quaking (QKI), a splicing factor induced during EMT, regulates a subset of events including the length of its own transcript. Analysis of QKI crosslinked immunoprecipitation (CLIP)-sequencing data identified the binding of QKI within 3' untranslated regions (UTRs) was enriched near cleavage and polyadenylation sites. Following QKI knockdown, APA of many transcripts is altered to produce predominantly shorter 3'UTRs associated with reduced gene expression. These findings reveal the changes in APA that occur during EMT and identify a potential role for QKI in this process.

Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation.

BACKGROUND: Triple-negative breast cancers (TNBC) have a relatively poor prognosis and responses to targeted therapies. Between 25 and 39% of TNBCs are claudin-low, a poorly differentiated subtype enriched for mesenchymal, stem cell and mitogen-activated signaling pathways. We investigated the role of the cell-surface co-receptor NRP1 in the biology of claudin-low TNBC. METHODS: The clinical prognostic value of NRP1 was determined by Kaplan-Meier analysis. GSVA analysis of METABRIC and Oslo2 transcriptomics datasets was used to correlate NRP1 expression with claudin-low gene signature scores. NRP1 siRNA knockdown was performed in MDA-MB-231, BT-549, SUM159 and Hs578T claudin-low cells and proliferation and viability measured by live cell imaging and DNA quantification. In SUM159 orthotopic xenograft models using NSG mice, NRP1 was suppressed by shRNA knockdown or systemic treatment with the NRP1-targeted monoclonal antibody Vesencumab. NRP1-mediated signaling pathways were interrogated by protein array and Western blotting. RESULTS: High NRP1 expression was associated with shorter relapse- and metastasis-free survival specifically in ER-negative BrCa cohorts. NRP1 was over-expressed specifically in claudin-low clinical samples and cell lines, and NRP1 knockdown reduced proliferation of claudin-low cells and prolonged survival in a claudin-low orthotopic xenograft model. NRP1 inhibition suppressed expression of the mesenchymal and stem cell markers ZEB1 and ITGA6, respectively, compromised spheroid-initiating capacity and exerted potent anti-tumor effects on claudin-low orthotopic xenografts (12.8-fold reduction in endpoint tumor volume). NRP1 was required to maintain maximal RAS/MAPK signaling via EGFR and PDGFR, a hallmark of claudin-low tumors. CONCLUSIONS: These data implicate NRP1 in the aggressive phenotype of claudin-low breast cancer and offer a novel targeted therapeutic approach to this poor prognosis subtype.

miR-200/375 control epithelial plasticity-associated alternative splicing by repressing the RNA-binding protein Quaking.

Members of the miR-200 family are critical gatekeepers of the epithelial state, restraining expression of pro-mesenchymal genes that drive epithelial-mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR-200c and another epithelial-enriched miRNA, miR-375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA-binding protein Quaking (QKI). During EMT, QKI-5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI-5 is both necessary and sufficient to direct EMT-associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial-derived cancer types. Importantly, several actin cytoskeleton-associated genes are directly targeted by both QKI and miR-200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT These findings demonstrate the existence of a miR-200/miR-375/QKI axis that impacts cancer-associated epithelial cell plasticity through widespread control of alternative splicing.

Differential subcellular and extracellular localisations of proteins required for insulin-like growth factor- and extracellular matrix-induced signalling events in breast cancer progression.

BACKGROUND: Cancer metastasis is the main contributor to breast cancer fatalities as women with the metastatic disease have poorer survival outcomes than women with localised breast cancers. There is an urgent need to develop appropriate prognostic methods to stratify patients based on the propensities of their cancers to metastasise. The insulin-like growth factor (IGF)-I: IGF binding protein (IGFBP):vitronectin complexes have been shown to stimulate changes in gene expression favouring increased breast cancer cell survival and a migratory phenotype. We therefore investigated the prognostic potential of these IGF- and extracellular matrix (ECM) interaction-induced proteins in the early identification of breast cancers with a propensity to metastasise using patient-derived tissue microarrays. METHODS: Semiquantitative immunohistochemistry analyses were performed to compare the extracellular and subcellular distribution of IGF- and ECM-induced signalling proteins among matched normal, primary cancer and metastatic cancer formalin-fixed paraffin-embedded breast tissue samples. RESULTS: The IGF- and ECM-induced signalling proteins were differentially expressed between subcellular and extracellular localisations. Vitronectin and IGFBP-5 immunoreactivity was lower while ß1 integrin immunoreactivity was higher in the stroma surrounding metastatic cancer tissues, as compared to normal breast and primary cancer stromal tissues. Similarly, immunoreactive stratifin was found to be increased in the stroma of primary as well as metastatic breast tissues. Immunoreactive fibronectin and ß1 integrin was found to be highly expressed at the leading edge of tumours. Based on the immunoreactivity it was apparent that the cell signalling proteins AKT1 and ERK1/2 shuffled from the nucleus to the cytoplasm with tumour progression. CONCLUSION: This is the first in-depth, compartmentalised analysis of the distribution of IGF- and ECM-induced signalling proteins in metastatic breast cancers. This study has provided insights into the changing pattern of cellular localisation and expression of IGF- and ECM-induced signalling proteins in different stages of breast cancer. The differential distribution of these biomarkers could provide important prognostic and predictive indicators that may assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy.

Quaking isoforms cooperate to promote the mesenchymal phenotype.

The RNA-binding protein Quaking (QKI) has widespread effects on mRNA regulation including alternative splicing, stability, translation, and localization of target mRNAs. Recently, QKI was found to be induced during epithelial-mesenchymal transition (EMT), where it promotes a mesenchymal alternative splicing signature that contributes to the mesenchymal phenotype. QKI is itself alternatively spliced to produce three major isoforms, QKI-5, QKI-6, and QKI-7. While QKI-5 is primarily localized to the nucleus where it controls mesenchymal splicing during EMT, the functions of the two predominantly cytoplasmic isoforms, QKI-6 and QKI-7, in this context remain uncharacterized. Here we used CRISPR-mediated depletion of QKI in a human mammary epithelial cell model of EMT and studied the effects of expressing the QKI isoforms in isolation and in combination. QKI-5 was required to induce mesenchymal morphology, while combined expression of QKI-5 with either QKI-6 or QKI-7 further enhanced mesenchymal morphology and cell migration. In addition, we found that QKI-6 and QKI-7 can partially localize to the nucleus and contribute to alternative splicing of QKI target genes. These findings indicate that the QKI isoforms function in a dynamic and cooperative manner to promote the mesenchymal phenotype.

Vitronectin--master controller or micromanager?

The concept that the mammalian glycoprotein vitronectin acts as a biological 'glue' and key controller of mammalian tissue repair and remodelling activity is emerging from nearly 50 years of experimental in vitro and in vivo data. Unexpectedly, the vitronectin-knockout (VN-KO) mouse was found to be viable and to have largely normal phenotype. However, diligent observation revealed that the VN-KO animal exhibits delayed coagulation and poor wound healing. This is interpreted to indicate that VN occupies a role in the earliest events of thrombogenesis and tissue repair. VN is the foundation upon which the thrombus grows in an organised structure. In addition to sealing the wound, the thrombus also serves to protect the underlying tissue from oxidation, is a reservoir of mitogens and tissue repair mediators, and provides a provisional scaffold for the repairing tissue. In the absence of VN (e.g., VN-KO animal), this cascade is disrupted before it begins. A wide variety of biologically active species associate with VN. Although initial studies were focused on mitogens, other classes of bioactives (e.g., glycosaminoglycans and metalloproteinases) are now also known to specifically interact with VN. Although some interactions are transient, others are long-lived and often result in multi-protein complexes. Multi-protein complexes provide several advantages: prolonging molecular interactions, sustaining local concentrations, facilitating co-stimulation of cell surface receptors and thereby enhancing cellular/biological responses. We contend that these, or equivalent, multi-protein complexes facilitate VN polyfunctionality in vivo. It is also likely that many of the species demonstrated to associate with VN in vitro, also associate with VN in vivo in similar multi-protein complexes. Thus, the predominant biological function of VN is that of a master controller of the extracellular environment; informing, and possibly instructing cells 'where' to behave, 'when' to behave and 'how' to behave (i.e., appropriately for the current circumstance).

Core epithelial-to-mesenchymal transition interactome gene-expression signature is associated with claudin-low and metaplastic breast cancer subtypes.

The epithelial-to-mesenchymal transition (EMT) produces cancer cells that are invasive, migratory, and exhibit stem cell characteristics, hallmarks of cells that have the potential to generate metastases. Inducers of the EMT include several transcription factors (TFs), such as Goosecoid, Snail, and Twist, as well as the secreted TGF-beta1. Each of these factors is capable, on its own, of inducing an EMT in the human mammary epithelial (HMLE) cell line. However, the interactions between these regulators are poorly understood. Overexpression of each of the above EMT inducers up-regulates a subset of other EMT-inducing TFs, with Twist, Zeb1, Zeb2, TGF-beta1, and FOXC2 being commonly induced. Up-regulation of Slug and FOXC2 by either Snail or Twist does not depend on TGF-beta1 signaling. Gene expression signatures (GESs) derived by overexpressing EMT-inducing TFs reveal that the Twist GES and Snail GES are the most similar, although the Goosecoid GES is the least similar to the others. An EMT core signature was derived from the changes in gene expression shared by up-regulation of Gsc, Snail, Twist, and TGF-beta1 and by down-regulation of E-cadherin, loss of which can also trigger an EMT in certain cell types. The EMT core signature associates closely with the claudin-low and metaplastic breast cancer subtypes and correlates negatively with pathological complete response. Additionally, the expression level of FOXC1, another EMT inducer, correlates strongly with poor survival of breast cancer patients.

Gene expression based inference of cancer drug sensitivity.

Inter and intra-tumoral heterogeneity are major stumbling blocks in the treatment of cancer and are responsible for imparting differential drug responses in cancer patients. Recently, the availability of high-throughput screening datasets has paved the way for machine learning based personalized therapy recommendations using the molecular profiles of cancer specimens. In this study, we introduce Precily, a predictive modeling approach to infer treatment response in cancers using gene expression data. In this context, we demonstrate the benefits of considering pathway activity estimates in tandem with drug descriptors as features. We apply Precily on single-cell and bulk RNA sequencing data associated with hundreds of cancer cell lines. We then assess the predictability of treatment outcomes using our in-house prostate cancer cell line and xenografts datasets exposed to differential treatment conditions. Further, we demonstrate the applicability of our approach on patient drug response data from The Cancer Genome Atlas and an independent clinical study describing the treatment journey of three melanoma patients. Our findings highlight the importance of chemo-transcriptomics approaches in cancer treatment selection.

Epithelial to Mesenchymal Transition Regulates Surface PD-L1 via CMTM6 and CMTM7 Induction in Breast Cancer.

CMTM6 is a critical regulator of cell surface expression of PD-L1 in tumor cells, but little is known about the transcriptional regulation of CMTM6. Here we report that the expression of CMTM6 positively correlates with the epithelial to mesenchymal transition (EMT) score in breast cancer cell lines and with the major EMT marker Vimentin in triple-negative breast cancers (TNBC). We showed that CMTM6 is concomitantly overexpressed with PD-L1 in breast mesenchymal compared with the epithelial cells. Driving a mesenchymal phenotype in SNAI1-inducible MCF-7 cells (MCF-7Mes cells) increased both PD-L1 and CMTM6. CMTM6 silencing in MCF-7Mes cells partially reduced cell surface expression of PD-L1, indicating that a proportion of the PD-L1 on the surface of MCF-7Mes cells depends on CMTM6. We also found a positive correlation between CMTM3 and CMTM7 expression with EMT score in breast cancer cells, and with Vimentin in TNBC patients. Dual knockdown of CMTM6 and CMTM7 significantly decreased PD-L1 surface expression in MCF-7Mes cells, indicating that both CMTM6 and CMTM7 regulate the expression of PD-L1. This study highlights the importance of CMTM6 and CMTM7 in EMT-induced PD-L1 and suggests that EMT, CMTM6 or CMTM7 modulators can be combined with anti-PD-L1 in patients with highly aggressive breast cancer.

Mechanistic investigations into interactions between IGF-I and IGFBPs and their impact on facilitating cell migration on vitronectin.

Numerous studies have reported links between insulin-like growth factors (IGFs) and the extra-cellular matrix protein vitronectin (VN). We ourselves have reported that IGF-I binds to VN via IGF-binding proteins (IGFBPs) to stimulate HaCaT and MCF-7 cell migration. Here, we detail the functional evaluation of IGFBP-1, -2, -3, -4 and -6 in the presence and absence of IGF-I and VN. The data presented here, combined with our prior data on IGFBP-5, suggest that IGFBP-3, -4 and -5 are the most effective at stimulating cell migration in combination with IGF-I and VN. In addition, we demonstrate that different regions within IGFBP-3 and -4 are critical for complex formation. Furthermore, we examine whether multi-protein complexes of IGF-I and IGFBPs associated with fibronectin and collagen IV are also able to enhance functional biological responses.

The epithelial-to-mesenchymal transition and cancer stem cells: a coalition against cancer therapies.

During cancer progression, some cells within the primary tumor may reactivate a latent embryonic program known as epithelial-to-mesenchymal transition (EMT). Through EMT, transformed epithelial cells can acquire the mesenchymal traits that seem to facilitate metastasis. Indeed, there is accumulating evidence that EMT and mesenchymal-related gene expression are associated with aggressive breast cancer subtypes and poor clinical outcome in breast cancer patients. More recently, the EMT program was shown to endow normal and transformed mammary epithelial cells with stem cell properties, including the ability to self-renew and efficiently initiate tumors. This link between EMT and stem cells may have numerous implications in the progression of breast tumors. The EMT process may facilitate the generation of cancer cells with the mesenchymal traits needed for dissemination as well as the self-renewal properties needed for initiation of secondary tumors. Breast cancer stem cells are resistant to many conventional cancer therapies, which can promote tumor relapse. Therefore, the generation of cancer stem cells by EMT may promote the development of refractory and resistant breast tumors. The purpose of this review is to summarize the findings related to EMT and stem cells in cancer progression and therapy resistance.

Transient Sox9 Expression Facilitates Resistance to Androgen-Targeted Therapy in Prostate Cancer.

PURPOSE: Patients with metastatic prostate cancer are increasingly presenting with treatment-resistant, androgen receptor-negative/low (AR-/Low) tumors, with or without neuroendocrine characteristics, in processes attributed to tumor cell plasticity. This plasticity has been modeled by Rb1/p53 knockdown/knockout and is accompanied by overexpression of the pluripotency factor, Sox2. Here, we explore the role of the developmental transcription factor Sox9 in the process of prostate cancer therapy response and tumor progression. EXPERIMENTAL DESIGN: Unique prostate cancer cell models that capture AR-/Low stem cell-like intermediates were analyzed for features of plasticity and the functional role of Sox9. Human prostate cancer xenografts and tissue microarrays were evaluated for temporal alterations in Sox9 expression. The role of NF-κB pathway activity in Sox9 overexpression was explored. RESULTS: Prostate cancer stem cell-like intermediates have reduced Rb1 and p53 protein expression and overexpress Sox2 as well as Sox9. Sox9 was required for spheroid growth, and overexpression increased invasiveness and neural features of prostate cancer cells. Sox9 was transiently upregulated in castration-induced progression of prostate cancer xenografts and was specifically overexpressed in neoadjuvant hormone therapy (NHT)-treated patient tumors. High Sox9 expression in NHT-treated patients predicts biochemical recurrence. Finally, we link Sox9 induction to NF-κB dimer activation in prostate cancer cells. CONCLUSIONS: Developmentally reprogrammed prostate cancer cell models recapitulate features of clinically advanced prostate tumors, including downregulated Rb1/p53 and overexpression of Sox2 with Sox9. Sox9 is a marker of a transitional state that identifies prostate cancer cells under the stress of therapeutic assault and facilitates progression to therapy resistance. Its expression may index the relative activity of the NF-κB pathway.

Insulin Enhances Migration and Invasion in Prostate Cancer Cells by Up-Regulation of FOXC2.

Androgen deprivation therapy (ADT) is the standard treatment for advanced prostate cancer (PCa), yet many patients relapse with lethal metastatic disease. With this loss of androgens, increased cell plasticity has been observed as an adaptive response to ADT. This includes gain of invasive and migratory capabilities, which may contribute to PCa metastasis. Hyperinsulinemia, which develops as a side-effect of ADT, has been associated with increased tumor aggressiveness and faster treatment failure. We investigated the direct effects of insulin in PCa cells that may contribute to this progression. We measured cell migration and invasion induced by insulin using wound healing and transwell assays in a range of PCa cell lines of variable androgen dependency (LNCaP, 22RV1, DuCaP, and DU145 cell lines). To determine the molecular events driving insulin-induced invasion we used transcriptomics, quantitative real time-PCR, and immunoblotting in three PCa cell lines. Insulin increased invasiveness of PCa cells, upregulating Forkhead Box Protein C2 (FOXC2), and activating key PCa cell plasticity mechanisms including gene changes consistent with epithelial-to-mesenchymal transition (EMT) and a neuroendocrine phenotype. Additionally, analysis of publicly available clinical PCa tumor data showed metastatic prostate tumors demonstrate a positive correlation between insulin receptor expression and the EMT transcription factor FOXC2. The insulin receptor is not suitable to target clinically however, our data shows that actions of insulin in PCa cells may be suppressed by inhibiting downstream signaling molecules, PI3K and ERK1/2. This study identifies for the first time, a mechanism for insulin-driven cancer cell motility and supports the concept that targeting insulin signaling at the level of the PCa tumor may extend the therapeutic efficacy of ADT.

A molecular portrait of epithelial-mesenchymal plasticity in prostate cancer associated with clinical outcome.

The propensity of cancer cells to transition between epithelial and mesenchymal phenotypic states via the epithelial-mesenchymal transition (EMT) program can regulate metastatic processes, cancer progression, and treatment resistance. Transcriptional investigations using reversible models of EMT, revealed the mesenchymal-to-epithelial reverting transition (MErT) to be enriched in clinical samples of metastatic castrate resistant prostate cancer (mCRPC). From this enrichment, a metastasis-derived gene signature was identified that predicted more rapid cancer relapse and reduced survival across multiple human carcinoma types. Additionally, the transcriptional profile of MErT is not a simple mirror image of EMT as tumour cells retain a transcriptional "memory" following a reversible EMT. This memory was also enriched in mCRPC samples. Cumulatively, our studies reveal the transcriptional profile of epithelial-mesenchymal plasticity and highlight the unique transcriptional properties of MErT. Furthermore, our findings provide evidence to support the association of epithelial plasticity with poor clinical outcomes in multiple human carcinoma types.

Correction: A molecular portrait of epithelial-mesenchymal plasticity in prostate cancer associated with clinical outcome.

Following the publication of the above article, the authors noted an error in Figure 4, panel B. The colours of the localized and mCRPC samples were accidentally switched. The authors have corrected the colour scheme and added a key to the figure. They have also updated the colour scheme of panel C, both bars are now red instead of one red and one blue. The authors wish to apologize for any inconvenience caused.

Substrate-bound insulin-like growth factor (IGF)-I-IGF binding protein-vitronectin-stimulated breast cell migration is enhanced by coactivation of the phosphatidylinositide 3-Kinase/AKT pathway by alphav-integrins and the IGF-I receptor.

IGF-I can bind to the extracellular matrix protein vitronectin (VN) through the involvement of IGF-binding proteins-2, -3, -4, and -5. Because IGF-I and VN have established roles in tumor cell dissemination, we were keen to investigate the functional consequences of the interaction of IGF-I, IGF binding proteins (IGFBPs), and VN in tumor cell biology. Hence, functional responses of MCF-7 breast carcinoma cells and normal nontumorgenic MCF-10A mammary epithelial cells were investigated to allow side-by-side comparisons of these complexes in both cancerous and normal breast cells. We demonstrate that substrate-bound IGF-I-IGFBP-VN complexes stimulate synergistic increases in cellular migration in both cell types. Studies using IGF-I analogs determined this stimulation to be dependent on both heterotrimeric IGF-I-IGFBP-VN complex formation and the involvement of the IGF-I receptor (IGF-IR). Furthermore, the enhanced cellular migration was abolished on incubation of MCF-7 and MCF-10A cells with function blocking antibodies directed at VN-binding integrins and the IGF-IR. Analysis of the signal transduction pathways underlying the enhanced cell migration revealed that the complexes stimulate a transient activation of the ERK/MAPK signaling pathway while simultaneously producing a sustained activation of the phosphatidylinositide 3-kinase/AKT pathway. Experiments using pharmacological inhibitors of these pathways determined a requirement for phosphatidylinositide 3-kinase/AKT activation in the observed response. Overexpression of wild type and activated AKT further increases substrate-bound IGF-I-IGFBP-VN-stimulated migration. This study provides the first mechanistic insights into the action of IGF-I-IGFBP-VN complexes and adds further evidence to support the involvement of VN-binding integrins and their cooperativity with the IGF-IR in the promotion of tumor cell migration.

Targeting Insulin-Like Growth Factor-I and Extracellular Matrix Interactions in Melanoma Progression.

Insulin-like growth factor (IGF)-I binds to the ECM protein vitronectin (VN) through IGF binding proteins (IGFBPs) to enhance proliferation and migration of skin keratinocytes and fibroblasts. Although evidence exists for the role of individual components of the complex (IGF-I, IGFBP-3 and VN), the cellular functions stimulated by these proteins together as a complex remains un-investigated in melanoma cells. We report here that the IGF-I:IGFBP-3:VN trimeric complex stimulates a dose-dependent increase in the proliferation and migration of WM35 and Sk-MEL28 melanoma cells. In 3D Matrigel™ and hydrogel cultures, both cell lines formed primary tumor-like spheroids, which increased in size in a dose-dependent manner in response to the trimeric complex. Furthermore, we reveal IGFBP-3:VN protein complexes in malignant melanoma and squamous cell carcinoma patient tissues, where the IGFBP-3:VN complex was seen to be predominantly tumor cell-associated. Peptide antagonists designed to target the binding of IGF-I:IGFBP-3 to VN were demonstrated to inhibit IGF-I:IGFBP-3:VN-stimulated cell migration, invasion and 3D tumor cell growth of melanoma cells. Overall, this study provides new data on IGF:ECM interactions in skin malignancies and demonstrates the potential usefulness of a growth factor:ECM-disrupting strategy for abrogating tumor progression.

Folate-targeted amphiphilic cyclodextrin nanoparticles incorporating a fusogenic peptide deliver therapeutic siRNA and inhibit the invasive capacity of 3D prostate cancer tumours.

The main barrier to the development of an effective RNA interference (RNAi) therapy is the lack of a suitable delivery vector. Modified cyclodextrins have emerged in recent years for the delivery of siRNA. In the present study, a folate-targeted amphiphilic cyclodextrin was formulated using DSPE-PEG5000-folate to target prostate cancer cells. The fusogenic peptide GALA was included in the formulation to aid in the endosomal release of siRNA. Targeted nanoparticles were less than 200nm in size with a neutral surface charge. The complexes were able to bind siRNA and protect it from serum nucleases. Incubation with excess free folate resulted in a significant decrease in the uptake of targeted nanoparticles in LNCaP and PC3 cells, both of which have been reported to have differing pathways of folate uptake. There was a significant reduction in the therapeutic targets, ZEB1 and NRP1 at mRNA and protein level following treatment with targeted complexes. In preliminary functional assays using 3D spheroids, treatment of PC3 tumours with targeted complexes with ZEB1 and NRP1 siRNA resulted in more compact colonies relative to the untargeted controls and inhibited infiltration into the Matrigel™ layer.

Therapy-induced developmental reprogramming of prostate cancer cells and acquired therapy resistance.

Treatment-induced neuroendocrine transdifferentiation (NEtD) complicates therapies for metastatic prostate cancer (PCa). Based on evidence that PCa cells can transdifferentiate to other neuroectodermally-derived cell lineages in vitro, we proposed that NEtD requires first an intermediary reprogramming to metastable cancer stem-like cells (CSCs) of a neural class and we demonstrate that several different AR+/PSA+ PCa cell lines were efficiently reprogrammed to, maintained and propagated as CSCs by growth in androgen-free neural/neural crest (N/NC) stem medium. Such reprogrammed cells lost features of prostate differentiation; gained features of N/NC stem cells and tumor-initiating potential; were resistant to androgen signaling inhibition; and acquired an invasive phenotype in vitro and in vivo. When placed back into serum-containing mediums, reprogrammed cells could be re-differentiated to N-/NC-derived cell lineages or return back to an AR+ prostate-like state. Once returned, the AR+ cells were resistant to androgen signaling inhibition. Acute androgen deprivation or anti-androgen treatment in serum-containing medium led to the transient appearance of a sub-population of cells with similar characteristics. Finally, a 132 gene signature derived from reprogrammed PCa cell lines distinguished tumors from PCa patients with adverse outcomes. This model may explain neural manifestations of PCa associated with lethal disease. The metastable nature of the reprogrammed stem-like PCa cells suggests that cycles of PCa cell reprogramming followed by re-differentiation may support disease progression and therapeutic resistance. The ability of a gene signature from reprogrammed PCa cells to identify tumors from patients with metastasis or PCa-specific mortality implies that developmental reprogramming is linked to aggressive tumor behaviors.

Repositioning "old" drugs for new causes: identifying new inhibitors of prostate cancer cell migration and invasion.

The majority of prostate cancer (PCa) deaths occur due to the metastatic spread of tumor cells to distant organs. Currently, there is a lack of effective therapies once tumor cells have spread outside the prostate. It is therefore imperative to rapidly develop therapeutics to inhibit the metastatic spread of tumor cells. Gain of cell motility and invasive properties is the first step of metastasis and by inhibiting motility one can potentially inhibit metastasis. Using the drug repositioning strategy, we developed a cell-based multi-parameter primary screening assay to identify drugs that inhibit the migratory and invasive properties of metastatic PC-3 PCa cells. Following the completion of the primary screening assay, 33 drugs were identified from an FDA approved drug library that either inhibited migration or were cytotoxic to the PC-3 cells. Based on the data obtained from the subsequent validation studies, mitoxantrone hydrochloride, simvastatin, fluvastatin and vandetanib were identified as strong candidates that can inhibit both the migration and invasion of PC-3 cells without significantly affecting cell viability. By employing the drug repositioning strategy instead of a de novo drug discovery and development strategy, the identified drug candidates have the potential to be rapidly translated into the clinic for the management of men with aggressive forms of PCa.