Faculty Bibliography

BACKGROUND: Recently there has been an increased interest in the role of tumor-associated stroma in prostate tumorigenesis, but little is known about the respective roles of stomal ERalpha and ERbeta in prostate cancer (PCa). This study characterizes the expression patterns of ERalpha and ERbeta in tumor-associated stroma in association with various clinicopathological factors of importance in PCa prognosis and treatment. DESIGN: Immunohistochemistry was performed using antibodies against ERalpha and ERbeta to characterize their expression patterns in PCa tissue. Stromal ER levels (ERalpha and ERbeta) on tissue sections (n=47), were compared between tumor associated stroma and adjacent benign associated stroma. Immunohistochemistry was also performed on a PCa tissue microarray (TMA) (n=177) to correlate stromal expression with various clinicopathological parameters. The levels of ER nuclear expression were scored semi-quantitatively. RESULTS: The expression levels of both ERalpha and ERbeta were significantly lower in tumor-associated stroma than stroma surrounding benign prostatic glands on the same tissue section (ERalpha: p<0.01; ERbeta: p=0.01). When correlated with clinicopathological factors, the level of ERalpha expression in tumor-associated stroma showed a positive correlation with Gleason score (R(2)=0.8638). The expression of ERalpha was higher in PCa with advanced tumor stage (p=0.05) and not significantly different in extraprostatic extension (p>0.05). The level of ERbeta expression in tumor-associated stroma was decreased in patients older than 60 years compared to younger patients (p=0.01). CONCLUSION: This study demonstrates significant down-regulation of ERalpha and ERbeta expression in the tumor-associated stroma of PCa. However, the level of ERalpha expression in tumor-associated stroma shows a positive correlation with cancer differentiation and tumor stage.

Global microRNA (miRNA) profile may predict prostate cancer (PCa) behaviors. In this study, we examined global miRNA expression by miRNA profiling as well as specific miRNA expression levels in PCa epithelium and stroma by in situ hybridization (ISH) and correlated with various clinicopathological features. We first performed comprehensive miRNA profiling on 27 macrodissected cases of PCa by miRNA microarray. A total of 299 miRNAs were significantly dysregulated in high grade and advanced stage PCa. We demonstrated that PCa can be readily classified into high grade/stage and low-grade/stage groups by its global miRNA expression profile. Next, we examined the expression of several selected dysregulated miRNAs, including let-7c, miR-21, miR-27a, miR-30c, and miR-219, in PCa by ISH. The levels of miRNA expression in epithelial and stromal cells were scored semiquantitatively and compared with clinicopathological features, including age, race, Gleason score, stage, PSA recurrence, metastasis, hormone resistance and survival. We found that the expression of miR-30c and miR-219 were significantly down-regulated in PCa. miR-21 and miR-30c were significantly down-regulated in PCa in African Americans compared to Caucasian Americans. In addition, down-regulation of let-7c, miR-21, miR-30c, and miR-219 are associated with metastatic disease. Furthermore, down-regulation of miR-30c and let-7c are significantly associated with androgen-dependent PCa. In PCa stromal cells, let-7c downregulation is significantly associated with extraprostatic extension. Our data suggest that selected miRNAs may serve as potential biomarkers to predict cancer progression.

Localized urinary tract amyloidosis (UTA) is a rare disease that mimics neoplasia clinically, cystoscopically, and radiologically. We report eleven cases of isolated UTA from the urinary bladder (n=7) and upper urinary tract including the ureter (n=2) and renal pelvis (n=2). All cases clinically presented as mass lesions prior to histologic examination and clinically suggested a neoplastic process. The amyloid composition in most cases was mixed Kappa and Lambda light chains. All cases were cured after surgical excision except one case which was diagnosed as plasmacytosis/plasmacytoma six months later. Localized amyloidosis of the urinary tract usually has a benign clinical course and simple resection is recommended after systemic disease is ruled out.

Transcriptional intermediary factor 1 gamma (Tif1gamma) (Ectodermin/PTC7/RFG7/TRIM33) is a transcriptional cofactor with an important role in the regulation of the TGFbeta pathway. It has been suggested that it competes with Smad2/Smad3 for binding to Smad4, or alternatively that it may target Smad4 for degradation, although its role in carcinogenesis is unclear. In this study, we showed that Tif1gamma interacts with Smad1/Smad4 complex in vivo, using both yeast two-hybrid and coimmunoprecipitation assays. We demonstrated that Tif1gamma inhibits transcriptional activity of the Smad1/Smad4 complex through its PHD domain or bromo-domainin pancreatic cells by luciferase assay. Additionally, there is a dynamic inverse relationship between the levels of Tif1gamma and Smad4 in benign and malignant pancreatic cell lines. Overexpression of Tif1gamma resulted in decreased level of Smad4. Both overexpression and knockdown of Tif1gamma resulted in growth inhibition in both benign and cancerous pancreatic cell lines, attributable to a G2-phase cell cycle arrest, but only knockdown of Tif1gamma reduces tumor cell invasiveness in vitro. Our study demonstrated that imbalanced expression of Tif1gamma results in inhibition of pancreatic ductal epithelial cell growth. In addition, knockdown of Tif1gamma may inhibit tumor invasion. These data suggest that Tif1gamma might serve as a potential therapeutic target for pancreatic cancer.

The degree of differentiation in human cancers generally reflects the degree of malignancy, with the most undifferentiated cancer being also the highest grade and the most aggressive. High-grade serous ovarian cancers (HGSOC) are poorly differentiated and fast-growing malignancies. The molecular mechanisms underlying the poor differentiation of HGSOC has not been completely characterized. Evidence suggests that microRNAs (miRs) are dysregulated in HGSOC. Therefore, we focused on those miRs that are relevant to tumor differentiation. Expression profiling of miRs in HGSOC, indicated miR-106a and its family members were significantly upregulated. Upregulation of miR-106a was further validated by real-time RT-PCR and miR in situ hybridization in a large cohort of HGSOC specimens. Overexpression of miR-106a in benign and malignant ovarian cells significantly increased the cellular proliferation rate and expanded the side-population fraction. In particular, SKOV3 cells with miR-106a overexpression had significantly higher tumor initial/stem cell population (CD24 and CD133 positive cells) than control SKOV3 cells. Among many miR-106a predicated target genes, p130 (RBL2), an RB tumor suppressor family member, was not only confirmed as a specific target of miR-106a but also related to tumor growth and differentiation. The importance of mir-106a and RBL2 was further demonstrated in vivo, in which, SKOV3 cells overexpressing miR-106a formed poorly differentiated carcinomas and had reduced RBL2 levels. To our knowledge, this is the first study of miR-106a mediating proliferation and tumor differentiation in HGSOC. Implications: The current study suggests that the RB tumor suppressor pathway is a critical regulator of growth and differentiation in HGSOC.

Bone morphogenetic protein (BMP) signaling has emerged as an important regulator of sensory neuron development. Using a three-generation forward genetic screen in mice we have identified Megf8 as a novel modifier of BMP4 signaling in trigeminal ganglion (TG) neurons. Loss of Megf8 disrupts axon guidance in the peripheral nervous system and leads to defects in development of the limb, heart, and left-right patterning, defects that resemble those observed in Bmp4 loss-of-function mice. Bmp4 is expressed in a pattern that defines the permissive field for the peripheral projections of TG axons and mice lacking BMP signaling in sensory neurons exhibit TG axon defects that resemble those observed in Megf8 (-/-) embryos. Furthermore, TG axon growth is robustly inhibited by BMP4 and this inhibition is dependent on Megf8. Thus, our data suggest that Megf8 is involved in mediating BMP4 signaling and guidance of developing TG axons. DOI:http://dx.doi.org/10.7554/eLife.01160.001.

Integration of cellular signaling pathways with androgen receptor (AR) signaling can be achieved through phosphorylation of AR by cellular kinases. However, the kinases responsible for phosphorylating the AR at numerous sites and the functional consequences of AR phosphorylation are only partially understood. Bioinformatic analysis revealed AR serine 213 (S213) as a putative substrate for PIM1, a kinase overexpressed in prostate cancer. Therefore, phosphorylation of AR serine 213 by PIM1 was examined using a phosphorylation site-specific antibody. Wild-type PIM1, but not catalytically inactive PIM1, specifically phosphorylated AR but not an AR serine-to-alanine mutant (S213A). In vitro kinase assays confirmed that PIM1 can phosphorylate AR S213 in a ligand-independent manner and cell type-specific phosphorylation was observed in prostate cancer cell lines. Upon PIM1 overexpression, AR phosphorylation was observed in the absence of hormone and was further increased in the presence of hormone in LNCaP, LNCaP-abl and VCaP cells. Moreover, phosphorylation of AR was reduced in the presence of PIM kinase inhibitors. An examination of AR-mediated transcription showed that reporter gene activity was reduced in the presence of PIM1 and wild-type AR, but not S213A mutant AR. Androgen-mediated transcription of endogenous PSA, Nkx3.1 and IGFBP5 was also decreased in the presence of PIM1, whereas IL6, cyclin A1 and caveolin 2 were increased. Immunohistochemical analysis of prostate cancer tissue microarrays showed significant P-AR S213 expression that was associated with hormone refractory prostate cancers, likely identifying cells with catalytically active PIM1. In addition, prostate cancers expressing a high level of P-AR S213 were twice as likely to be from biochemically recurrent cancers. Thus, AR phosphorylation by PIM1 at S213 impacts gene transcription and is highly prevalent in aggressive prostate cancer.Oncogene advance online publication, 17 September 2012; doi:10.1038/onc.2012.412.

Mouse models of prostate cancer (PCa) are critical for understanding the biology of PCa initiation, progression, and treatment modalities. Here, we summarize recent advances in PCa mouse models that led to new insights into specific gene functions in PCa. For example, the study of transgenic mice with TMPRSS2/ERG, an androgen regulated fusion protein, revealed its role in developing PCa precursor lesions, prostate intraepithelial neoplasia (PIN) but not sufficient for PCa development. Double deficiency of Pten and Smad4 leads to a high incidence of metastatic PCa. Targeted deletion of Pten in castration-resistant Nkx3-1-expressing cells (CARNs) results in rapid carcinoma formation after androgen-mediated regeneration, indicating progenitor cells with luminal characteristics can play a role in initiation of PCa. Transgenic mice with activated oncogenes, growth factors, and steroid hormone receptors or inactivated tumor suppressors continue to provide insight for disease progression from initiation to metastasis. Further development of new PCa models with spatial and temporal regulation of candidate gene expression will likely enhance our understanding of the complex events that lead to PCa initiation and progression, thereby invoking novel strategies to combat this common disease in men.

BACKGROUND: Androgen receptor (AR) expression in breast cancers may serve as a prognostic and predictive marker. We examined the expression pattern of AR and its phosphorylated forms, Ser-213 (AR-Ser[P]-213) and Ser-650 (AR-Ser[P]-650), in breast cancer and evaluated their association with clinicopathological parameters. METHODS: Immunohistochemistry was performed on primary and distant metastatic breast cancers and benign breast tissue using antibodies against AR, AR-Ser(P)-213, and AR-Ser(P)-650. The levels of cytoplasmic and nuclear expression were scored semiquantitatively using a histoscore. RESULTS: Nuclear staining of AR was observed in all benign breast tissue and 67% of cancer cases. Nuclear and cytoplasmic AR-Ser(P)-213 was increased in breast cancers 2-fold (P = .0014) and 1.7-fold (P = .05), respectively, compared with benign controls, whereas nuclear and cytoplasmic AR-Ser(P)-650 expression was decreased in tumors by 1.9-fold and 1.7-fold (both P < .0001), respectively. Increased expression of nuclear or cytoplasmic AR-Ser(P)-213 was observed in metastatic breast cancers (1.3-fold, P = .05), ER-negative (2.6-fold, P = .001), and invasive ductal carcinoma (6.8-fold, P = .04). AR-Ser(P)-650 expression was downregulated in lymph node-positive breast cancers (1.4-fold, P = .02) but was upregulated in invasive ductal carcinomas (3.2-fold, P < .0001) and metastases (1.5-fold, P = .003). Moreover, in ER-negative breast cancers, nuclear AR-Ser(P)-650 was decreased (1.4-fold, P = .005), and cytoplasmic AR-Ser(P)-650 was increased (1.4-fold, P = .003). CONCLUSIONS: AR and its phosphorylation at serines 213 and 650 are differentially expressed in breast cancer tumorigenesis and progression. Phosphorylation of AR at serines 213 and 650 is increased in ER-negative breast cancers, ductal carcinomas, and metastases and may have predictive value in breast cancer prognosis. Cancer 2013;000:000-000. (c) 2013 American Cancer Society.

AIM: To develop and validate a technique for construction of intermediate density tissue microarray (TMA) slides based on the transfer of tissue from pre-existing routine slides provided for pathology diagnosis with validation to show preservation of morphology and antigenicity of the transferred tissue. METHODS: A prostate cancer TMA was constructed using 20 cores from radical prostatectomy slides. This technique entails removal of the coverslip on each slide and reinforcement of the tissue by covering with Mount-Quick liquid mounting medium. The attached tissue with its new scaffold is 'biopsied' using a TMA needle (1.5 mm in diameter). The resultant biopsy disc is then transferred onto a recipient slide, with adhesion of the disc to the slide accomplished using heavy pressure. The preservation of morphology and antigenicity of this TMA is tested in comparison to a traditional TMA. RESULTS: After immunohistochemical staining, 35 of 39 cores (89.7%) on the patch TMA were intact compared with 39 of 40 cores (97.5%) on the traditional TMA (p>0.1). Expression patterns and density of the antigens (34betaE12, p63 and AMACR) on the patch TMA were almost identical to the traditional TMA. CONCLUSIONS: Patch TMA represents a viable alternative for tissue-based immunohistochemistry studies when paraffin blocks are unavailable. This may be a valuable tool for allowing the use of archival slide material for immunohistochemistry and enabling a standardised TMA platform to be used when the slides sent for review from other institutions are the only source of tissue available.