Chun-Peng Liao

Pten null prostate tumorigenesis and AKT activation are blocked by targeted knockout of ER chaperone GRP78/BiP in prostate epithelium

GRP78/BiP has recently emerged as a novel biomarker for aggressive prostate cancer. Here, we report that homozygous deletion of Grp78 specifically in mouse prostate epithelium suppresses prostate tumorigenesis without affecting postnatal prostate development and growth. Mouse prostates with double conditional knockout of Grp78 and Pten exhibit normal histology and cytology, in contrast to the invasive adenocarcinoma in mouse prostates with Pten inactivation. AKT activation in Pten null prostate epithelium is inhibited by Grp78 homozygous deletion, corresponding with suppression of AKT phosphorylation by GRP78 knockdown in prostate cancer cell line. Thus, inactivation of GRP78 may represent a previously undescribed approach to stop prostate cancer and potentially other cancers resulting from the loss of PTEN tumor suppression and/or activation of the oncogenic AKT.

Cancer-associated fibroblasts enhance the gland-forming capability of prostate cancer stem cells.

Signals originating from cancer-associated fibroblasts (CAF) may positively regulate proliferation and tumorigenicity in prostate cancer. In this study, we investigated whether CAFs may regulate the biology of prostate cancer stem cells (CSC) by using a conditional Pten deletion mouse model of prostate adenocarcinoma to isolate both CAF cultures and CSC-enriched cell fractions from the tumors. CSCs that were isolated possessed self-renewal, spheroid-forming, and multipotential differentiation activities in tissue culture, segregating with a cell fraction exhibiting a signature expression phenotype, including SCA-1 (high), CD49f (high), CK5 (high), p63 (high), Survivin (high), RUNX2 (high), CD44 (low), CD133 (low), CK18 (low), and Androgen Receptor (low). CSC spheroid-forming efficiency was differentially influenced by the nature of fibroblasts in a coculture system: Compared with mouse urogenital sinus mesenchyme or normal prostate fibroblasts, CAFs enhanced spheroid formation, with the spheroids displaying generally larger sizes and more complex histology. Graft experiments showed that CSCs admixed with CAFs produced prostatic glandular structures with more numerous lesions, high proliferative index, and tumor-like histopathologies, compared with those formed in the presence of normal prostate fibroblasts. Together, our findings underscore a significant role of CAFs in CSC biology.

Mouse Models of Prostate Adenocarcinoma with the Capacity to Monitor Spontaneous Carcinogenesis by Bioluminescence or Fluorescence

The application of Cre/loxP technology has resulted in a new generation of conditional mouse models of prostate cancer. Here, we describe the improvement of the conditional Pten deletion model of prostate adenocarcinoma by combining it with either a conditional luciferase or enhanced green fluorescent protein reporter line. In these models, the recombination mechanism that inactivates the Pten alleles also activates the reporter gene. In the luciferase reporter model, the growth of the primary cancer can be followed noninvasively by bioluminescence imaging (BLI). Surgical castration of tumor-bearing animals leads to a reduced bioluminescence signal corresponding to tumor regression that is verified at necropsy. When castrated animals are maintained, the emergence of androgen depletion-independent cancer is detected using BLI at times varying from 7 to 28 weeks postcastration. The ability to monitor growth, regression, or relapse of the tumor with the use of BLI lead to the collection of tumors at different stages of development. By comparing the distribution of phenotypically distinct populations of epithelial cells in cancer tissues, we noted that the degree of hyperplasia of cells with neuroendocrine differentiation significantly increases in the recurrent cancer relative to the primary cancer, a characteristic which may parallel the appearance of a neuroendocrine phenotype in human androgen depletion-independent cancer. The enhanced green fluorescent protein model, at necropsy, can provide an opportunity to locate or assess tumor volume or to isolate enriched populations of cancer cells from tumor tissues via fluorescence-based technologies. These refined models should be useful in the elucidation of mechanisms of prostate cancer progression, and for the development of approaches to preclinical intervention.

Cooperation between FGF8b Overexpression and PTEN Deficiency in Prostate Tumorigenesis

Two commonly occurring genetic aberrations of human prostate cancer [i.e., overexpression of a mitogenic polypeptide (fibroblast growth factor 8, isoform b or FGF8b) and loss of function of PTEN tumor suppressor] were recapitulated into a new combinatorial mouse model. This model harboring the Fgf8b transgene and haploinsufficiency in Pten, both in a prostate epithelium-specific manner, yielded prostatic adenocarcinoma with readily detectable lymph node metastases, whereas single models with each of the defects were shown earlier to progress generally only up to prostatic intraepithelial neoplasia (PIN). In addition to late age-related development of typical adenocarcinoma, the model also displayed a low incidence of mucinous adenocarcinoma, a rare variant type of human prostatic adenocarcinoma. The cooperation between FGF8b activation and PTEN deficiency must be linked to acquisition of additional genetic alterations for the progression of the lesions to primary adenocarcinoma. Here, we identified loss of heterozygosity at the Pten gene leading to bialleic loss, as a necessary secondary event, indicating that a complete loss of PTEN function is required in the development of invasive cancer in the model. Analyses of expression of downstream mediators phospho-AKT (p-AKT) and p27(KIP1), in various types of lesions, however, revealed a complex picture. Although PIN lesions displayed relatively strong expression of p-AKT and p27(KIP1), there was a notable heterogeneity with variable decrease in their immunostaining in adenocarcinomas. Together, the results further underscore the notion that besides activation of AKT by loss of PTEN function, other PTEN-regulated pathways must be operative for progression of lesions from PIN to adenocarcinoma.

Monoamine oxidase A mediates prostate tumorigenesis and cancer metastasis

Tumors from patients with high-grade aggressive prostate cancer (PCa) exhibit increased expression of monoamine oxidase A (MAOA), a mitochondrial enzyme that degrades monoamine neurotransmitters and dietary amines. Despite the association between MAOA and aggressive PCa, it is unclear how MAOA promotes PCa progression. Here, we found that MAOA functions to induce epithelial-to-mesenchymal transition (EMT) and stabilize the transcription factor HIF1α, which mediates hypoxia through an elevation of ROS, thus enhancing growth, invasiveness, and metastasis of PCa cells. Knockdown and overexpression of MAOA in human PCa cell lines indicated that MAOA induces EMT through activation of VEGF and its coreceptor neuropilin-1. MAOA-dependent activation of neuropilin-1 promoted AKT/FOXO1/TWIST1 signaling, allowing FOXO1 binding at the TWIST1 promoter. Importantly, the MAOA-dependent HIF1α/VEGF-A/FOXO1/TWIST1 pathway was activated in high-grade PCa specimens, and knockdown of MAOA reduced or even eliminated prostate tumor growth and metastasis in PCa xenograft mouse models. Pharmacological inhibition of MAOA activity also reduced PCa xenograft growth in mice. Moreover, high MAOA expression in PCa tissues correlated with worse clinical outcomes in PCa patients. These findings collectively characterize the contribution of MAOA in PCa pathogenesis and suggest that MAOA has potential as a therapeutic target in PCa.

TPL2/COT/MAP3K8 (TPL2) Activation Promotes Androgen Depletion-Independent (ADI) Prostate Cancer Growth

Background Despite its initial positive response to hormone ablation therapy, prostate cancers invariably recur in more aggressive, treatment resistant forms. The lack of our understanding of underlying genetic alterations for the transition from androgen-dependent (AD) to ADI prostate cancer growth hampers our ability to develop target-driven therapeutic strategies for the efficient treatment of ADI prostate cancer. Methodology/Principal Findings By screening a library of activated human kinases, we have identified TPL2, encoding a serine/threonine kinase, as driving ADI prostate cancer growth. TPL2 activation by over-expressing either wild-type or a constitutively activated form of TPL2 induced ADI growth, whereas the suppression of TPL2 expression and its kinase activity in ADI prostate cancer cells inhibited cell proliferation under androgen-depleted conditions. Most importantly, TPL2 is upregulated in ADI prostate cancers of both the Pten deletion mouse model and the clinical prostate cancer specimens. Conclusions/Significance Together these data suggest that TPL2 kinase plays a critical role in the promotion of ADI prostate cancer progression. Furthermore, the suppression of TPL2 diminishes ADI prostate cancer growth and a high frequency of TPL2 overexpression in human ADI prostate cancer samples validates TPL2 as a target for the treatment of this deadly disease.

CAF-Secreted Annexin A1 Induces Prostate Cancer Cells to Gain Stem Cell-like Features

Annexin A1 (AnxA1), a phospholipid-binding protein and regulator of glucocorticoid-induced inflammatory signaling, has implications in cancer. Here, a role for AnxA1 in prostate adenocarcinoma was determined using primary cultures and a tumor cell line (cE1), all derived from the conditional Pten deletion mouse model of prostate cancer. AnxA1 secretion by prostate-derived cancer-associated fibroblasts (CAF) was significantly higher than by normal prostate fibroblasts (NPF). Prostate tumor cells were sorted to enrich for epithelial subpopulations based on nonhematopoietic lineage, high SCA-1, and high or medium levels of CD49f. Compared with controls, AnxA1 enhanced stem cell–like properties in high- and medium-expression subpopulations of sorted cE1 and primary cells, in vitro, through formation of greater number of spheroids with increased complexity, and in vivo, through generation of more, larger, and histologically complex glandular structures, along with increased expression of p63, a basal/progenitor marker. The differentiated medium-expression subpopulations from cE1 and primary cells were most susceptible to gain stem cell–like properties as shown by increased spheroid and glandular formation. Further supporting this increased plasticity, AnxA1 was shown to regulate epithelial-to-mesenchymal transition in cE1 cells. These results suggest that CAF-secreted AnxA1 contributes to tumor stem cell dynamics via two separate but complementary pathways: induction of a dedifferentiation process leading to generation of stem-like cells from a subpopulation of cancer epithelial cells and stimulation of proliferation and differentiation of the cancer stem-like cells. Implications: AnxA1 participates in a paradigm in which malignant prostate epithelial cells that are not cancer stem cells are induced to gain cancer stem cell–like properties. Mol Cancer Res; 12(4); 607–21. ©2014 AACR.

Cancer Stem Cells and Microenvironment in Prostate Cancer Progression

For a study of interactions between the cancer-associated fibroblasts (CAFs) and the putative prostate cancer stem cells (CSCs), we used a conditional Pten deletion mouse model of prostatic adenocarcinoma to isolate both CAF cultures and CSC-enriched cell fractions from the primary tumors. The CSC subpopulation exhibited a collective phenotype of Lin−/SCA-1hi/CD49fhi/p63hi/CK5hi/ARlo/CK18lo/Survivinhi/Runx2hi and contained cells with the ability to both self-renew and differentiate into basal and luminal cells in vitro. The spheroids generated from the CSC-enriched subpopulation mimicked the glandular structures that could be produced from a similarly isolated cell fraction from the normal mouse prostate. The efficiency of spheroid formation was found to be influenced differentially by the nature of the fibroblasts that were co-cultured in the 3-D system. The growth and differentiation properties of the CSCs were significantly more enhanced by factors released from CAFs relative to normal prostate fibroblasts (NPFs). Additionally, increased commitment to differentiation to the luminal cell lineage was noted when CAFs were present. When CSCs admixed with either CAFs or NPFs were examined for formation of prostatic glandular structures in renal grafts in vivo, the lesions formed were generally more in numbers in the presence of CAFs than NPFs. Furthermore, lesions formed with CAFs often displayed tumor-like complex histopathology and contained increased numbers of proliferating cells. Taken together, the results suggested that the CAFs in the prostate tumor microenvironment can contribute to the biologic properties of the CSCs and by this account may play a major role in prostate tumorigenesis and progression. Thus, it would be important now to identify the paracrine and/or juxtacrine factors that are responsible for the stimulation of the cancer stem cells.

Dependence of Castration-Resistant Prostate Cancer (CRPC) Stem Cells on CRPC-Associated Fibroblasts

We previously established a role for cancer‐associated fibroblasts (CAF) in enhancing the self‐renewal and differentiation potentials of putative prostate cancer stem cells (CSC). Our published work focused on androgen‐dependent prostate cancer (ADPC) using the conditional Pten deletion mouse model. Employing the same model, we now describe the interaction of CAF and CSC in castration‐resistant prostate cancer (CRPC). CAF isolated from ADPC (ADPCAF) and from CRPC (CRPCAF) were compared in terms of their ability to support organoid formation and tumor initiation by CSC from CRPC (CRPCSC) in vitro and in vivo. CRPCSC formed spheroids in vitro and well‐differentiated glandular structures under the renal capsules of recipient mice in vivo more effectively in the presence of CRPCAF compared to ADPCAF. Furthermore, whereas CSC with CAF from ADPC formed mostly well‐differentiated tumors in our previous study, we now show that CRPCSC, when combined with CRPCAF (but not ADPCAF), can form aggressive, poorly‐differentiated tumors. The potential of CRPCAF to support organoid/tumor formation by CRPCSC remained greater even when compared to 10‐fold more ADPCAF, suggesting that paracrine factors produced specifically by CRPCAF preferentially potentiate the stemness and tumorigenic properties of the corresponding CSC. This apparently unique property of CRPCAF was notable when the CAF and CSC were grafted in either intact or castrated recipient mice. In both environments, CRPCAF induced in the epithelial compartment higher proliferative activity compared to ADPCAF, indicated by a higher Ki67 index. Factors released by CRPCAF to regulate CRPCSC may be targeted to develop novel therapeutic approaches to manage advanced prostate cancer. J. Cell. Physiol. 229: 1170–1176, 2014.

Expression and Functional Role of Orphan Receptor GPR158 in Prostate Cancer Growth and Progression

Prostate cancer (PCa) is the second-leading cause of cancer-related mortality, after lung cancer, in men from developed countries. In its early stages, primary tumor growth is dependent on androgens, thus generally can be controlled by androgen deprivation therapy (ADT). Eventually however, the disease progresses to castration-resistant prostate cancer (CRPC), a lethal form in need of more effective treatments. G-protein coupled receptors (GPCRs) comprise a large clan of cell surface proteins that have been implicated as therapeutic targets in PCa growth and progression. The findings reported here provide intriguing evidence of a role for the newly characterized glutamate family member GPR158 in PCa growth and progression. We found that GPR158 promotes PCa cell proliferation independent of androgen receptor (AR) functionality and that this requires its localization in the nucleus of the cell. This suggests that GPR158 acts by mechanisms different from other GPCRs. GPR158 expression is stimulated by androgens and GPR158 stimulates AR expression, implying a potential to sensitize tumors to low androgen conditions during ADT via a positive feedback loop. Further, we found GPR158 expression correlates with a neuroendocrine (NE) differentiation phenotype and promotes anchorage-independent colony formation implying a role for GPR158 in therapeutic progression and tumor formation. GPR158 expression was increased at the invading front of prostate tumors that formed in the genetically defined conditional Pten knockout mouse model, and co-localized with elevated AR expression in the cell nucleus. Kaplan-Meier analysis on a dataset from the Memorial Sloan Kettering cancer genome portal showed that increased GPR158 expression in tumors is associated with lower disease-free survival. Our findings strongly suggest that pharmaceuticals targeting GPR158 activities could represent a novel and innovative approach to the prevention and management of CRPC.