Hong Pu

Targeting TGF-β in prostate cancer: therapeutic possibilities during tumor progression

Background: TGF-β regulates prostate growth by inhibiting epithelial cell proliferation and inducing apoptosis through eliciting a dynamic signaling pathway. In metastatic prostate cancer, however, TGF-β serves as a tumor promoter. TGF-β engages Smad-dependent and Smad-independent mechanisms to exert its action. During prostate tumorigenesis, prostate cells exhibit loss or mutation of TGF-β transmembrane receptors. Increased production of TGF-β causes immunosuppression, extracellular matrix degradation, epithelia to mesenchymal transition and angiogenesis that promotes tumor cell invasion and metastasis. Objective: The molecular basis for effective therapeutic targeting of TGF-β must be directed towards the double-edge-sword nature of the cytokine: Inhibiting the TGF-β tumor promoter capabilities in advanced metastatic prostate cancer, although retaining the growth-inhibitory abilities exhibited in early stages of prostate tumorigenesis. Results/conclusion: The current understanding of the therapeutic possibilities of targeting TGF-β signaling during prostate tumor progression is built on preclinical studies. Studies targeting TGF-β signaling pathway for the treatment of several human malignancies include the use of neutralizing antibodies, antisense oligonucelotides and small molecule inhibitors of kinase activity of the receptor complex. This review focuses on exploiting the therapeutic potential of targeting TGF-β signaling in the context of its contribution to prostate cancer initiation and progression to metastasis.

Dysfunctional Transforming Growth Factor-β Receptor II Accelerates Prostate Tumorigenesis in the TRAMP Mouse Model

The contribution of a dysfunctional transforming growth factor-beta type II receptor (TGF beta RII) to prostate cancer initiation and progression was investigated in an in vivo mouse model. Transgenic mice harboring the dominant-negative mutant TGF-beta type II receptor (DNTGF beta RII) in mouse epithelial cell were crossed with the TRAMP prostate cancer transgenic mouse to characterize the in vivo consequences of inactivated TGF-beta signaling on prostate tumor initiation and progression. Histopathologic diagnosis of prostate specimens from the TRAMP+/DNTGF beta RII double transgenic mice revealed the appearance of early malignant changes and subsequently highly aggressive prostate tumors at a younger age, compared with littermates TRAMP+/Wt TGF beta RII mice. Immunohistochemical and Western blotting analysis revealed significantly increased proliferative and apoptotic activities, as well as vascularity and macrophage infiltration that correlated with an elevated vascular endothelial growth factor and MCP-1 protein levels in prostates from TRAMP+/DNTGF beta RII+ mice. An epithelial-mesenchymal transition (EMT) effect was also detected in prostates of TRAMP+/DNTGF beta RII mice, as documented by the loss of epithelial markers (E-cadherin and beta-catenin) and up-regulation of mesenchymal markers (N-cadherin) and EMT-transcription factor Snail. A significant increase in the androgen receptor mRNA and protein levels was associated with the early onset of prostate tumorigenesis in TRAMP+/DNTGF beta RII mice. Our results indicate that in vivo disruption of TGF-beta signaling accelerates the pathologic malignant changes in the prostate by altering the kinetics of prostate growth and inducing EMT. The study also suggests that a dysfunctional TGF beta RII augments androgen receptor expression and promotes inflammation in early stage tumor growth, thus conferring a significant contribution by TGF-beta to prostate cancer progression.

Cofilin drives cell-invasive and metastatic responses to TGF-β in prostate cancer.

Cofilin (CFL) is an F-actin-severing protein required for the cytoskeleton reorganization and filopodia formation, which drives cell migration. CFL binding and severing of F-actin is controlled by Ser3 phosphorylation, but the contributions of this step to cell migration during invasion and metastasis of cancer cells are unclear. In this study, we addressed the question in prostate cancer cells, including the response to TGF-β, a critical regulator of migration. In cells expressing wild-type CFL, TGF-β treatment increased LIMK-2 activity and cofilin phosphorylation, decreasing filopodia formation. Conversely, constitutively active CFL (SerAla) promoted filipodia formation and cell migration mediated by TGF-β. Notably, in cocultures of prostate cancer epithelial cells and cancer-associated fibroblasts, active CFL promoted invasive migration in response to TGF-β in the microenvironment. Further, constitutively active CFL elevated the metastatic ability of prostate cancer cells in vivo. We found that levels of active CFL correlated with metastasis in a mouse model of prostate tumor and that in human prostate cancer, CFL expression was increased significantly in metastatic tumors. Our findings show that the actin-severing protein CFL coordinates responses to TGF-β that are needed for invasive cancer migration and metastasis.

Multinucleation and Mesenchymal-to-Epithelial Transition Alleviate Resistance to Combined Cabazitaxel and Antiandrogen Therapy in Advanced Prostate Cancer

Patients with metastatic castration-resistant prostate cancer (CRPC) frequently develop therapeutic resistance to taxane chemotherapy and antiandrogens. Cabazitaxel is a second-line taxane chemotherapeutic agent that provides additional survival benefits to patients with advanced disease. In this study, we sought to identify the mechanism of action of combined cabazitaxel and androgen receptor (AR) targeting in preclinical models of advanced prostate cancer. We found that cabazitaxel induced mitotic spindle collapse and multinucleation by targeting the microtubule depolymerizing kinesins and inhibiting AR. In androgen-responsive tumors, treatment with the AR inhibitor, enzalutamide, overcame resistance to cabazitaxel. Combination treatment of human CRPC xenografts with cabazitaxel and enzalutamide reversed epithelial-mesenchymal transition (EMT) to mesenchymal-epithelial transition (MET) and led to multinucleation, while retaining nuclear AR. In a transgenic mouse model of androgen-responsive prostate cancer, cabazitaxel treatment induced MET, glandular redifferentiation, and AR nuclear localization that was inhibited by androgen deprivation. Collectively, our preclinical studies demonstrate that prostate tumor resistance to cabazitaxel can be overcome by antiandrogen-mediated EMT-MET cycling in androgen-sensitive tumors but not in CRPC. Moreover, AR splice variants may preclude patients with advanced disease from responding to cabazitaxel chemotherapy and antiandrogen combination therapy. This evidence enables a significant insight into therapeutic cross-resistance to taxane chemotherapy and androgen deprivation therapy in advanced prostate cancer.

Autophagy and oxidative stress in gliomas with IDH1 mutations

IDH1 mutations in gliomas associate with longer survival. Prooxidant and antiproliferative effects of IDH1 mutations and its d-2-hydroxyglutarate (2-HG) product have been described in vitro, but inconsistently observed. It is also unclear whether overexpression of mutant IDH1 in wild-type cells accurately phenocopies the effects of endogenous IDH1-mutations on tumor apoptosis and autophagy. Herein we investigated the effects of 2-HG and mutant IDH1 overexpression on proliferation, apoptosis, oxidative stress, and autophagy in IDH1 wild-type glioma cells, and compared those results with patient-derived tumors. 2-HG reduced viability and proliferation of U87MG and LN18 cells, triggered apoptosis in LN18 cells, and autophagy in U87MG cells. In vitro studies and flank xenografts of U87MG cells overexpressing R132H IDH1 exhibited increased oxidative stress, including increases of both manganese superoxide dismutase (MnSOD) and p62. Patient-derived IDH1-mutant tumors showed no significant differences in apoptosis or autophagy, but showed p62 accumulation and actually trended toward reduced MnSOD expression. These data indicate that mutant IDH1 and 2-HG can induce oxidative stress, autophagy, and apoptosis, but these effects vary greatly according to cell type.

PARP-1 regulates epithelial-mesenchymal transition (EMT) in prostate tumorigenesis.

Poly (ADP-ribose) polymerase (PARP) is involved in key cellular processes such as DNA replication and repair, gene transcription, cell proliferation and apoptosis. The role of PARP-1 in prostate cancer development and progression is not fully understood. The present study investigated the function of PARP-1 in prostate growth and tumorigenesis in vivo. Functional inactivation of PARP-1 by gene-targeted deletion led to a significant reduction in the prostate gland size in young PARP-1-/- mice (6 weeks) compared with wild-type (WT) littermates. To determine the effect of PARP-1 functional loss on prostate cancer onset, PARP-1-/- mice were crossed with the transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. Pathological assessment of prostate tumors revealed that TRAMP+/-, PARP-1-/- mice exhibited higher grade prostate tumors compared with TRAMP+/- PARP-1+/+ (16-28 weeks) that was associated with a significantly increased proliferative index and decreased apoptosis among the epithelial cells in TRAMP+/- PARP-1-/- prostate tumors. Furthermore tumors harboring PARP-1 loss, exhibited a downregulation of nuclear androgen receptor. Impairing PARP-1 led to increased levels of transforming growth factor-β (TGF-β) and Smads that correlated with induction of epithelial-mesenchymal transition (EMT), as established by loss of E-cadherin and β-catenin and upregulation of N-cadherin and ZEB-1. Our findings suggest that impaired PARP-1 function promotes prostate tumorigenesis in vivo via TGF-β-induced EMT. Defining the EMT control by PARP-1 during prostate cancer progression is of translational significance for optimizing PARP-1 therapeutic targeting and predicting response in metastatic castration-resistant prostate cancer.

Aberrant TGF-β signaling drives castration-resistant prostate cancer in a male mouse model of prostate tumorigenesis

The androgen receptor (AR) plays a critical role as a driver of castration-resistant prostate cancer (CRPC). Our previous studies demonstrated that disruption of transforming growth factor-β (TGF-β) signaling via introduction of dominant-negative transforming growth factor-β type II receptor (DNTGFβRII) in the prostate epithelium of transgenic adenocarcinoma of the prostate mice accelerated tumor. This study investigated the consequences of disrupted TGF-β signaling on prostate tumor growth under conditions of castration-induced androgen deprivation in the preclinical model of DNTGFβRII. Our results indicate that in response to androgen deprivation therapy (ADT) the proliferative index in prostate tumors from DNTGFβRII mice was higher compared with prostate tumors from TGFβRII wild-type (WT) mice, whereas there was a reduced incidence of apoptosis in tumors from DNTGFβRII. Protein and gene expression profiling revealed that tumors from DNTGFβRII mice exhibit a strong nuclear AR localization among the prostate tumor epithelial cells and increased AR messenger RNA after ADT. In contrast, TGFβRII WT mice exhibited a marked loss in nuclear AR in prostate tumor acini (20 weeks), followed by a downregulation of AR and transmembrane protease serine 2 messenger RNA. There was a significant increase in nuclear AR and activity in prostate tumors from castrate DNTGFβRII compared with TGFβRII WT mice. Consequential to aberrant TGF-β signaling, ADT enhanced expression and nuclear localization of Smad4 and β-catenin. Our findings support that under castrate conditions, aberrant TGF-β signaling leads to AR activation and β-catenin nuclear localization, an adaptation mechanism contributing to emergence of CRPC. The work defines a potentially significant new targeting platform for overcoming therapeutic resistance in CRPC.

TGF-β receptor I inhibitor enhances response to enzalutamide in a pre-clinical model of advanced prostate cancer

Prostate cancer progression is navigated by the androgen receptor (AR) and transforming‐growth factor‐β (TGF‐β) signaling. We previously demonstrated that aberrant TGF‐β signaling accelerates prostate tumor progression in a transgenic mouse model of prostate cancer via effects on epithelial‐mesenchymal transition (EMT), driving castration‐resistant prostate cancer (CRPC).

Predictive value of epithelial‐mesenchymal‐transition (EMT) signature and PARP‐1 in prostate cancer radioresistance

Epithelial‐mesenchymal‐transition (EMT) has been previously identified as a contributor to prostate cancer progression to metastasis and therapeutic resistance to antiandrogens and radiotherapy. In this study we conducted a retrospective analysis to investigate the significance of radiation‐induced EMT and consequential changes to the tumor microenvironment in biochemical recurrence and response to radiotherapy in prostate cancer patients.

Abstract 2567: Cabazitaxel chemotherapy targets mitotic kinesins resulting in multi-nucleation of prostate cancer cells: A novel mechanism of cross-resistance with antiandrogens in advanced CRPC

Progression to castration-resistant prostate cancer (CRPC) is characterized by increased androgen receptor (AR) and activated AR signaling despite castrate levels of androgens. The FDA-approved next generation microtubule stabilizing taxane, Cabazitaxel (CBZ) and novel anti-androgen Enzalutamide (MDV) have demonstrated additional survival benefits for patients with advanced CRPC. The present study pursued the mechanism of therapeutic resistance to CBZ and antiandrogen treatment in models of CRPC. The effect CBZ and/or MDV was examined in human prostate cancer cells: androgen independent (DU145 and PC3), androgen responsive (LNCaP and VCaP) and CRPC (22Rv1 and PC3-ARv567, harboring AR variants). Cell viability assessment revealed that CRPC PC3-ARv567es and the androgen-sensitive VCaP cells exhibited resistance to CBZ; however the 22Rv1 cells responded to CBZ and MDV combination with a significant increase in growth (P