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PDF file - 343KB, Marker Validation of Differentiated Cells.

PDF file - 123KB, K14 loss is coincidental with integrin a6 loss.

PDF file - 134KB, Constitutive Myc expression leads to cell death after differentiation.

The molecular mechanisms of luminal cell differentiation are not understood well enough to determine how differentiation goes awry during oncogenesis. Using RNA-Seq analysis, we discovered that CREB1 plays a central role in maintaining new luminal cell survival and that oncogenesis dramatically changes the CREB1-induced transcriptome. CREB1 is active in luminal cells, but not basal cells. We identified ING4 and its E3 ligase, JFK, as CREB1 transcriptional targets in luminal cells. During luminal cell differentiation, transient induction of ING4 expression is followed by a peak in CREB1 activity, while JFK increases concomitantly with CREB1 activation. Transient expression of ING4 is required for luminal cell induction; however, failure to properly down-regulate ING4 leads to luminal cell death. Consequently, blocking CREB1 increased ING4 expression, suppressed JFK, and led to luminal cell death. Thus, CREB1 is responsible for the suppression of ING4 required for luminal cell survival and maintenance. Oncogenic transformation by suppressing PTEN resulted in constitutive activation of CREB1. However, the tumor cells could no longer fully differentiate into luminal cells, failed to express ING4, and displayed a unique CREB1 transcriptome. Blocking CREB1 in tumorigenic cells suppressed tumor growth in vivo, rescued ING4 expression, and restored luminal cell formation, but ultimately induced luminal cell death. IHC of primary prostate tumors demonstrated a strong correlation between loss of ING4 and loss of PTEN. This is the first study to define a molecular mechanism whereby oncogenic loss of PTEN, leading to aberrant CREB1 activation, suppresses ING4 expression causing disruption of luminal cell differentiation.

Prostate cancer metastases primarily localize in the bone where they induce a unique osteoblastic response. Elevated Notch activity is associated with high-grade disease and metastasis. To address how Notch affects prostate cancer bone lesions, we manipulated Notch expression in mouse tibia xenografts and monitored tumor growth, lesion phenotype, and the bone microenvironment. Prostate cancer cell lines that induce mixed osteoblastic lesions in bone expressed 5-6 times more Notch3, than tumor cells that produce osteolytic lesions. Expression of active Notch3 (NICD3) in osteolytic tumors reduced osteolytic lesion area and enhanced osteoblastogenesis, while loss of Notch3 in osteoblastic tumors enhanced osteolytic lesion area and decreased osteoblastogensis. This was accompanied by a respective decrease and increase in the number of active osteoclasts and osteoblasts at the tumor-bone interface, without any effect on tumor proliferation. Conditioned medium from NICD3-expressing cells enhanced osteoblast differentiation and proliferation in vitro, while simultaneously inhibiting osteoclastogenesis. MMP-3 was specifically elevated and secreted by NICD3-expressing tumors, and inhibition of MMP-3 rescued the NICD3-induced osteoblastic phenotypes. Clinical osteoblastic bone metastasis samples had higher levels of Notch3 and MMP-3 compared to patient matched visceral metastases or osteolytic metastasis samples. We identified a Notch3-MMP-3 axis in human prostate cancer bone metastases that contributes to osteoblastic lesion formation by blocking osteoclast differentiation, while also contributing to osteoblastogenesis. These studies define a new role for Notch3 in manipulating the tumor microenvironment in bone metastases.

Cancers with homology-directed DNA repair (HRR) deficiency exhibit high response rates to poly(ADP-ribose) polymerase inhibitors (PARPi) and platinum chemotherapy. Though mutations disrupting BRCA1 and BRCA2 associate with HRR deficiency (HRRd), patterns of genomic aberrations and mutation signatures may be more sensitive and specific indicators of compromised repair. Here, we evaluated whole-exome sequences from 418 metastatic prostate cancers (mPCs) and determined that one-fifth exhibited genomic characteristics of HRRd that included Catalogue Of Somatic Mutations In Cancer mutation signature 3. Notably, a substantial fraction of tumors with genomic features of HRRd lacked biallelic loss of a core HRR-associated gene, such as BRCA2. In this subset, HRRd associated with loss of chromodomain helicase DNA binding protein 1 but not with mutations in serine-protein kinase ATM, cyclin dependent kinase 12, or checkpoint kinase 2. HRRd genomic status was strongly correlated with responses to PARPi and platinum chemotherapy, a finding that supports evaluating biomarkers reflecting functional HRRd for treatment allocation.

Prostate cancer (PC) is driven by androgen receptor (AR) activity, a master regulator of prostate development and homeostasis. Frontline therapies for metastatic PC deprive the AR of the activating ligands testosterone (T) and dihydrotestosterone (DHT) by limiting their biosynthesis or blocking AR binding. Notably, AR signaling is dichotomous, inducing growth at lower activity levels, while suppressing growth at higher levels. Recent clinical studies have exploited this effect by administration of supraphysiological concentrations of T, resulting in clinical responses and improvements in quality of life. However, the use of T as a therapeutic agent in oncology is limited by poor drug-like properties as well as rapid and variable metabolism. Here, we investigated the antitumor effects of selective AR modulators (SARMs), which are small-molecule nonsteroidal AR agonists developed to treat muscle wasting and cachexia. Several orally administered SARMs activated the AR program in PC models. AR cistromes regulated by steroidal androgens and SARMs were superimposable. Coregulatory proteins including HOXB13 and GRHL2 comprised AR complexes assembled by both androgens and SARMs. At bioavailable concentrations, SARMs repressed MYC oncoprotein expression and inhibited the growth of castration-sensitive and castration-resistant PC in vitro and in vivo. These results support further clinical investigation of SARMs for treating advanced PC.

The prostate is a walnut-sized gland that surrounds the urethra of males at the base of the bladder comprising a muscular portion, which controls the release of urine, and a glandular portion, which secretes fluids that nourish and protect sperms. Here, we report the development of a microfluidic-based model of a human prostate gland. The polydimethylsiloxane (PDMS) microfluidic device, consisting of two stacked microchannels separated by a polyester porous membrane, enables long-term in vitro cocultivation of human epithelial and stromal cells. The porous separation membrane provides an anchoring scaffold for long-term culturing of the two cell types on its opposite surfaces allowing paracrine signaling but not cell crossing between the two channels. The microfluidic device is transparent enabling high resolution bright-field and fluorescence imaging. Within this coculture model of a human epithelium/stroma interface, we simulated the functional development of the in vivo human prostate gland. We observed the successful differentiation of basal epithelial cells into luminal secretory cells determined biochemically by immunostaining with known differentiation biomarkers, particularly androgen receptor expression. We also observed morphological changes where glandlike mounds appeared with relatively empty centers reminiscent of prostatic glandular acini structures. This prostate-on-a-chip will facilitate the direct evaluation of paracrine and endocrine cross talk between these two cell types as well as studies associated with normal vs disease-related events such as prostate cancer.

Prostate cancer (PC) initially depends on androgen receptor (AR) signaling for survival and growth. Therapeutics designed to suppress AR activity serve as the primary intervention for advanced disease. However, supraphysiological androgen (SPA) concentrations can produce paradoxical responses leading to PC growth inhibition. We sought to discern the mechanisms by which SPA inhibits PC and to determine if molecular context associates with antitumor activity. SPA produced an AR-mediated, dose-dependent induction of DNA double-strand breaks, G(0)/G(1) cell-cycle arrest, and cellular senescence. SPA repressed genes involved in DNA repair and delayed the restoration of damaged DNA, which was augmented by poly (ADP-ribose) polymerase 1 inhibition. SPA-induced double-strand breaks were accentuated in BRCA2-deficient patients with PC, and combining SPA with poly (ADP-ribose) polymerase or DNA-dependent protein kinase inhibition further repressed growth. Next-generation sequencing was performed on biospecimens from patients with PC receiving SPA as part of ongoing phase II clinical trials. Patients with mutations in genes mediating homology-directed DNA repair were more likely to exhibit clinical responses to SPA. These results provide a mechanistic rationale for directing SPA therapy to patients with PC who have AR amplification or DNA repair deficiency and for combining SPA therapy with poly (ADP-ribose) polymerase inhibition.

A Microfluidic-based model of a human prostate gland has been developed featuring a 3D co-culture of epithelial and stromal cells. The model was used to investigate the effects of normal stroma on normal luminal epithelial cell differentiation, and to measure the ability of cancerous epithelium to convert normal stroma to cancer stroma. The ability of PIM1 kinase to induce cancer stroma was also tested in the model. The results demonstrate a promising potential of the model for cancer research applications.

Prostate cancer (PCa) is a disease of mutated and misregulated genes. However, primary prostate tumors have relatively few mutations, and only three genes (ERG,PTEN, andSPOP) are recurrently mutated in more than 10% of primary tumors. On the other hand, metastatic castration-resistant tumors have more mutations, but, with the exception of the androgen receptor gene (AR), no single gene is altered in more than half of tumors. Structural genomic rearrangements are common, includingERGfusions, copy gains involving theMYClocus, and copy losses containingPTEN. Overall, instead of being associated with a single dominant driver event, prostate tumors display various combinations of modifications in oncogenes and tumor suppressors. This review takes a broad look at the recent advances in PCa research, including understanding the genetic alterations that drive the disease and how specific mutations can sensitize tumors to potential therapies. We begin with an overview of the genomic landscape of primary and metastatic PCa, enabled by recent large-scale sequencing efforts. Advances in three-dimensional cell culture techniques and mouse models for PCa are also discussed, and particular emphasis is placed on the benefits of patient-derived xenograft models. We also review research into understanding how ETS fusions (in particular,TMPRSS2-ERG) andSPOPmutations contribute to tumor initiation. Next, we examine the recent findings on the prevalence of germline DNA repair mutations in about 12% of patients with metastatic disease and their potential benefit from the use of poly(ADP-ribose) polymerase (PARP) inhibitors and immune modulation. Lastly, we discuss the recent increased prevalence of AR-negative tumors (neuroendocrine and double-negative) and the current state of immunotherapy in PCa. AR remains the primary clinical target for PCa therapies; however, it does not act alone, and better understanding of supporting mutations may help guide the development of novel therapeutic strategies.

The mechanisms by which Myc overexpression or Pten loss promotes prostate cancer development are poorly understood. We identified the chromatin remodeling protein, ING4, as a crucial switch downstream of Myc and Pten that is required for human prostate epithelial differentiation. Myc-induced transient expression of ING4 is required for the differentiation of basal epithelial cells into luminal cells, while sustained ING4 expression induces apoptosis. ING4 expression is lost in >60% of human primary prostate tumors. ING4 or Pten loss prevents epithelial cell differentiation, which was necessary for tumorigenesis. Pten loss prevents differentiation by blocking ING4 expression, which is rescued by ING4 re-expression. Pten or ING4 loss generates tumor cells that co-express basal and luminal markers, indicating prostate oncogenesis occurs through disruption of an intermediate step in the prostate epithelial differentiation program. Thus, we identified a new epithelial cell differentiation switch involving Myc, Pten, and ING4, which when disrupted leads to prostate tumorigenesis. Myc overexpression and Pten loss are common genetic abnormalities in prostate cancer, whereas loss of the tumor suppressor ING4 has not been reported. This is the first demonstration that transient ING4 expression is absolutely required for epithelial differentiation, its expression is dependent on Myc and Pten, and it is lost in the majority of human prostate cancers. This is the first demonstration that loss of ING4, either directly or indirectly through loss of Pten, promotes Myc-driven oncogenesis by deregulating differentiation. The clinical implication is that Pten/ING4 negative and ING4-only negative tumors may reflect two distinct subtypes of prostate cancer. Cancer Res; 74(12); 3357–68. ©2014 AACR.

Background Short hairpin RNA (shRNA) is an established and effective tool for stable knock down of gene expression. Lentiviral vectors can be used to deliver shRNAs, thereby providing the ability to infect most mammalian cell types with high efficiency, regardless of proliferation state. Furthermore, the use of inducible promoters to drive shRNA expression allows for more thorough investigations into the specific timing of gene function in a variety of cellular processes. Moreover, inducible knockdown allows the investigation of genes that would be lethal or otherwise poorly tolerated if constitutively knocked down. Lentiviral inducible shRNA vectors are readily available, but unfortunately the process of cloning, screening, and testing shRNAs can be time-consuming and expensive. Therefore, we sought to refine a popular vector (Tet-pLKO-Puro) and streamline the cloning process with efficient protocols so that researchers can more efficiently utilize this powerful tool. Methods First, we modified the Tet-pLKO-Puro vector to make it easy (“EZ”) for molecular cloning (EZ-Tet-pLKO-Puro). Our primary modification was to shrink the stuffer region, which allows vector purification via polyethylene glycol precipitation thereby avoiding the need to purify DNA through agarose. In addition, we generated EZ-Tet-pLKO vectors with hygromycin or blasticidin resistance to provide greater flexibility in cell line engineering. Furthermore, we provide a detailed guide for utilizing these vectors, including shRNA design strategy and simplified screening methods. Results Notably, we emphasize the importance of loop sequence design and demonstrate that the addition of a single mismatch in the loop stem can greatly improve shRNA efficiency. Lastly, we display the robustness of the system with a doxycycline titration and recovery time course and provide a cost/benefit analysis comparing our system with purchasing pre-designed shRNA vectors. Conclusions Our aim was twofold: first, to take a very useful shRNA vector and make it more amenable for molecular cloning and, secondly, to provide a streamlined protocol and rationale for cost-effective design, cloning, and screening of shRNAs. With this knowledge, anyone can take advantage of this powerful tool to inducibly knockdown any gene of their choosing.

Many genes aberrantly expressed in prostate cancer are involved in normal basal to luminal cell differentiation. We previously demonstrated that transient ING4 expression is required for luminal cell differentiation and is downregulated in ~60% of primary prostate tumors. We further demonstrated in a primary prostate cancer model overexpressing ERG, MYC, and shPTEN (EMP) that loss of PTEN was responsible for ING4 loss. Furthermore, half of the human tumor samples that lose ING4 have also lost PTEN. However, we did not know how PTEN loss inhibits ING4 expression. Utilizing our in vitro differentiation model, whereby prostate basal epithelial cells (iPrEC) treated with KGF and androgen induce a suprabasal layer of luminal-like cells, and RNA-seq we identified transcriptional nodes required for luminal differentiation. Differentially expressed genes were analyzed by GeneGo to identify enriched transcription-factor signatures. Of the ~600 differentially regulated genes during differentiation, the largest signature (29% of genes) was CREB/ATF targets. Induction of Blimp1, Claudin1, and Plk2 and inhibition of Chek1 were further validated by qRT-PCR and immunoblotting. CREB/ATF bind constitutively to open chromatin CRE elements in the promoters of genes and are activated through signaling-induced phosphorylation at Ser133 by kinases, including AKT. We found that both CREB1 and ATF1 are inducibly phosphorylated midway through luminal differentiation, with ATF1 preceding CREB1. Knockdown of CREB1 with shRNA increased ING4, accelerated differentiation, and induced premature luminal cell death. Conversely, knockdown of ATF1 blocked ING4 induction and prevented suprabasal layer formation. CREB1/ATF1 ChIP was enriched at the ING4 promoter at mid-differentiation, when ING4 expression peaks. Additionally, CREB1/ATF1 was constitutively bound to the promoter of JFK, an E3-ligase that targets ING4 and whose mRNA levels increase during differentiation. Thus, we propose that ATF1 is required to induce ING4 transcription, while CREB1 suppresses ING4 and simultaneously activates its E3-ligase to tightly control the timing of ING4 expression. We compared the gene signature of luminal cells to that of the tumorigenic EMP cells and, surprisingly, found that 30% of the differentially expressed genes were also CREB/ATF targets. However, there is less than 10% overlap in these targets, indicating that CREB/ATF control distinct subsets of genes in differentiated luminal cells versus cancer cells. Some of EMP-specific CREB/ATF targets included GATA2, TWIST1, Necdin, and PPM1F, which were further validated by qRT-PCR and immunoblotting. CREB1 and ATF1 were highly phosphorylated in EMP cells and knockdown of CREB1 restored ING4 expression and suprabasal formation. Our working model is that AKT activation upon PTEN loss in transiently differentiating luminal cells results in premature and constitutive activation of CREB1/ATF1 bound to genes prior to induction of the ING4 chromatin switch. This prevents ING4 induction and the chromatin rearrangements required for terminal differentiation. In normal PrECs, CREB/ATF1 activation is tightly controlled by as yet undetermined factors and is only permitted when the proper CRE binding sites are exposed. This model helps to explain how loss of PTEN disrupts luminal cell terminal differentiation to promote prostate cancer oncogenesis. Citation Format: McLane Watson, Penny Berger, Sander Frank, Mary Winn, Cindy Miranti. CREB1 and ATF1 differentially regulate terminal prostate luminal differentiation by controlling the timing of ING4 expression, while CREB1 prevents ING4 expression upon PTEN loss in 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 B031.

This study identifies a new mechanism by which the receptor tyrosine kinase Met promotes cell survival. The ectodomain and transmembrane domain of Met, independently of kinase activity, are required to maintain integrin α3β1 on the cell surface to prevent activation of intrinsic and extrinsic cell death pathways and maintain autophagic flux., Matrix adhesion via integrins is required for cell survival. Adhesion of epithelial cells to laminin via integrin α3β1 was previously shown to activate at least two independent survival pathways. First, integrin α3β1 is required for autophagy-induced cell survival after growth factor deprivation. Second, integrin α3β1 independently activates two receptor tyrosine kinases, EGFR and Met, in the absence of ligands. EGFR signaling to Erk promotes survival independently of autophagy. To determine how Met promotes cell survival, we inhibited Met kinase activity or blocked its expression with RNA interference. Loss of Met expression, but not inhibition of Met kinase activity, induced apoptosis by reducing integrin α3β1 levels, activating anoikis, and blocking autophagy. Met was specifically required for the assembly of autophagosomes downstream of LC3II processing. Reexpression of wild-type Met, kinase-dead Met, or integrin α3 was sufficient to rescue death upon removal of endogenous Met. Integrin α3β1 coprecipitated and colocalized with Met in cells. The extracellular and transmembrane domain of Met was required to fully rescue cell death and restore integrin α3 expression. Thus Met promotes survival of laminin-adherent cells by maintaining integrin α3β1 via a kinase-independent mechanism.

Many pathways dysregulated in prostate cancer are also involved in epithelial differentiation. To better understand prostate tumor initiation, we sought to investigate specific genes and mechanisms required for normal basal to luminal cell differentiation. Utilizing human prostate basal epithelial cells and an in vitro differentiation model, we tested the hypothesis that regulation of NOTCH3 by the p38 MAPK family (hereafter p38-MAPK), via MYC, is required for luminal differentiation. Inhibition (SB202190 and BIRB796) or knockdown of p38α (also known as MAPK14) and/or p38δ (also known as MAPK13) prevented proper differentiation. Additionally, treatment with a γ-secretase inhibitor (RO4929097) or knockdown of NOTCH1 and/or NOTCH3 greatly impaired differentiation and caused luminal cell death. Constitutive p38-MAPK activation through MKK6(CA) increased NOTCH3 (but not NOTCH1) mRNA and protein levels, which was diminished upon MYC inhibition (10058-F4 and JQ1) or knockdown. Furthermore, we validated two NOTCH3 enhancer elements through a combination of enhancer (e)RNA detection (BruUV-seq) and luciferase reporter assays. Finally, we found that the NOTCH3 mRNA half-life increased during differentiation or upon acute p38-MAPK activation. These results reveal a new connection between p38-MAPK, MYC and NOTCH signaling, demonstrate two mechanisms of NOTCH3 regulation and provide evidence for NOTCH3 involvement in prostate luminal cell differentiation.

A major problem in the prostate cancer field is the inability to distinguish aggressive from indolent disease. This gap in knowledge stems from the fact that the mechanisms which trigger tumorigenesis in the prostate are poorly understood. While specific known genetic alterations are very common in prostate tumors, including Myc upregulation, fusion of Ets genes to androgen-regulated promoters, and loss of Pten, the specific roles of these aberrations in prostate tumor initiation and progression are poorly understood. Likewise, the cell of origin for prostate cancer remains controversial and may be linked to the aggressive potential of the tumor. One important clue about the oncogenic cell of origin is that prostate tumors coexpress basal and luminal protein markers normally restricted to their respective cell types in the prostate epithelia. We hypothesize this coexpression is indicative of the fact that prostate tumors arise as a defect in normal epithelial differentiation. Moreover, we hypothesize that rare bipotent cells in the prostate, which can give rise to basal or luminal cells, are particularly sensitive to oncogenic conversion and are a cell of origin for prostate cancer. In support of this hypothesis, many of the pathways involved in prostate differentiation are linked to genes commonly altered in prostate cancer. In this article, we review what is known about important differentiation pathways in the prostate (Myc, p38MAPK, Notch, PI3K/Pten) and how their misregulation in a transiently differentiating bipotent epithelial cell could lead to oncogenesis. Better understanding of normal prostate differentiation will offer new insights into tumor initiation and may help to explain the functional significance of common genetic alterations observed in prostate cancer. Additionally, this understanding could lead to new methods for classifying prostate tumors based on their differentiation status and may aid in identifying more aggressive tumors.

Myc is overexpressed in the majority of human prostate cancers and is a known determinant of cell fate, yet the cell of origin from which prostate cancers arise is controversial. Furthermore, the mechanisms by which oncogenes such as Myc disrupt prostate epithelial cell fate are poorly understood. Using a novel human in vitro differentiation model in which prostate basal epithelial cells are induced to differentiate into lumenal cells, we previously demonstrated that Myc-driven prostate cancer develops in an intermediate progenitor cell population whose full differentiation is derailed upon oncogenic transformation (Berger et al, Cancer Res 74:3357-68, 2014). In basal prostate epithelial cells, Myc is required for transient expression of the chromatin-binding protein ING4, which is required for lumenal cell differentiation. In human tissues, ING4 expression is lost in >60% of primary prostate tumors. Loss of ING4 prevented differentiation and was necessary for Myc-dependent tumorigenesis in vivo. ING4 loss generated Myc-dependent tumor cells co-expressing basal and lumenal markers, indicating Myc-dependent oncogenesis disrupted an intermediate step in the prostate epithelial differentiation program. Our objective for this study was to further elucidate the mechanisms by which Myc controls prostate epithelial cell fate. Myc is a known downstream target of Notch1, and several studies suggest Notch signaling is aberrant in prostate cancer; although the mechanistic details are vague. We found that Notch3 is required for lumenal cell differentiation and hypothesized that Notch3 expression is directly controlled by Myc. Inhibition of total Notch signaling with a γ-secretase inhibitor (RO4929097) prevented differentiation. Total Notch1 mRNA and protein levels change very little during differentiation; whereas both Notch3 mRNA and protein increase dramatically. Knock-down of Notch3 by shRNA blocked differentiation, while over expression of active Notch3 (NCID3) induced spontaneous differentiation. Less than 15% of the increase in Notch3 mRNA was attributable to increased mRNA stability, and required new protein synthesis. Temporally, Myc mRNA and protein levels increase prior to Notch3. Blocking Myc expression prevented Notch3 induction. The 2kb proximal promoter region of Notch3 lacked the elements that promote Notch3 induction. We identified a Notch3 enhancer element with Myc binding motifs that support differentiation-induced luciferase reporter activity. We further determined that p38α-MAPK is required for Myc and Notch3 induction. We are currently determining how Myc-dependent regulation of Notch3 influences Myc-dependent regulation of ING4. Thus, our studies demonstrate that at least 2 targets of Myc, ING4 and Notch3, control prostate epithelial cell fate, and that disruption of at least one of them is required for Myc-driven human prostate cancer development. In depth understanding of Myc-driven differentiation pathways will provide new insights into how oncogenic transformation by Myc in intermediate progenitor prostate epithelial cells gives rise to prostate cancer. Funding was provided by the Association for International Cancer Research, NIH/NCI CA154835, Department of Defense W81XWH-14-10479, and the Van Andel Research Institute. Citation Format: Sander B. Frank, Penny L. Berger, Cindy K. Miranti. Myc governs a prostate epithelial differentiation program involving chromatin remodeling protein ING4 and Notch3: Disruption of which is necessary for human prostate cancer development. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B18.