Janice M. Lage

Systemic distribution, subcellular localization and differential expression of sphingosine-1-phosphate receptors in benign and malignant human tissues.

AIMS Five sphingosine-1-phosphate receptors (S1PR): S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5 (S1PR1-5) have been shown to be involved in the proliferation and progression of various cancers. However, none of the S1PRs have been systemically investigated. In this study, we performed immunohistochemistry (IHC) for S1PR1-S1PR5 on different tissues, in order to simultaneously determine the systemic distribution, subcellular localization and expression level of all five S1PRs. METHODS We constructed tissue microarrays (TMAs) from 384 formalin-fixed paraffin-embedded (FFPE) blocks containing 183 benign and 201 malignant tissues from 34 human organs/systems. Then we performed IHC for all five S1PRs simultaneously on these TMA slides. The distribution, subcellular localization and expression of each S1PR were determined for each tissue. The data in benign and malignant tissues from the same organ/tissue were then compared using the Students t-test. In order to reconfirm the subcellular localization of each S1PR as determined by IHC, immunocytochemistry (ICC) was performed on several malignant cell lines. RESULTS We found that all five S1PRs are widely distributed in multiple human organs/systems. All S1PRs are expressed in both the cytoplasm and nucleus, except S1PR3, whose IHC signals are only seen in the nucleus. Interestingly, the S1PRs are rarely expressed on cellular membranes. Each S1PR is unique in its organ distribution, subcellular localization and expression level in benign and malignant tissues. Among the five S1PRs, S1PR5 has the highest expression level (in either the nucleus or cytoplasm), with S1PR1, 3, 2 and 4 following in descending order. Strong nuclear expression was seen for S1PR1, S1PR3 and S1PR5, whereas S1PR2 and S1PR4 show only weak staining. Four organs/tissues (adrenal gland, liver, brain and colon) show significant differences in IHC scores for the multiple S1PRs (nuclear and/or cytoplasmic), nine (stomach, lymphoid tissues, lung, ovary, cervix, pancreas, skin, soft tissues and uterus) show differences for only one S1PR (cytoplasmic or nuclear), and twenty three organs/tissues show no significant difference in IHC scores for any S1PR (cytoplasmic or nuclear) between benign and malignant changes. CONCLUSION This is the first study to evaluate the expression level of all S1PRs in benign and malignant tissues from multiple human organs. This study provides data regarding the systemic distribution, subcellular localization and differences in expression of all five S1PRs in benign and malignant changes for each organ/tissue.

Dietary pterostilbene is a novel MTA1-targeted chemopreventive and therapeutic agent in prostate cancer

Overexpression of the epigenetic modifier metastasis-associated protein 1 (MTA1) is associated with aggressive human prostate cancer. The purpose of this study was to determine MTA1- targeted chemopreventive and therapeutic efficacy of pterostilbene, a natural potent analog of resveratrol, in pre-clinical models of prostate cancer. Here, we show that high levels of MTA1 expression in Pten-loss prostate cooperate with key oncogenes, including c-Myc and Akt among others, to promote prostate cancer progression. Loss-of-function studies using human prostate cancer cells indicated direct involvement of MTA1 in inducing inflammation and epithelial-to-mesenchymal transition. Importantly, pharmacological inhibition of MTA1 by pterostilbene resulted in decreased proliferation and angiogenesis and increased apoptosis. This restrained prostatic intraepithelial neoplasia (PIN) formation in prostate-specific Pten heterozygous mice and reduced tumor development and progression in prostate-specific Pten-null mice. Our findings highlight MTA1 as a key upstream regulator of prostate tumorigenesis and cancer progression. More significantly, it offers pre-clinical proof for pterostilbene as a promising lead natural agent for MTA1-targeted chemopreventive and therapeutic strategy to curb prostate cancer.

Epigenetic potential of resveratrol and analogs in preclinical models of prostate cancer

Lifestyle, particularly diet, is a risk factor for prostate cancer. Dietary polyphenols such as resveratrol possess anticancer properties and therefore have chemopreventive and therapeutic potential. Resveratrol has pleiotropic effects, exerting its biological activity through multiple pathways and targets, including those associated with cancer. Numerous studies have demonstrated the anticancer effects of resveratrol and, to a lesser extent, its analogs, in tissue culture, while in vivo observations are limited. Here, we provide a concise summary of our results on epigenetic mechanisms of resveratrol and analogs mediated through regulation of chromatin modifier metastasis‐associated protein 1 (MTA1) and microRNAs (miRNAs), and highlight the anticancer effects of these compounds in preclinical models of prostate cancer. We suggest that the identified stilbene responsive mechanism–based biomarkers, such as MTA1 and oncogenic miRNAs, may become indicative of treatment efficacy in prostate cancer. Resveratrol analogs with better bioavailability, conferring superior pharmacological potencies and greater anticancer effects, may become stronger candidates for clinical development.

Geminin overexpression promotes imatinib sensitive breast cancer: a novel treatment approach for aggressive breast cancers, including a subset of triple negative.

Breast cancer is the second leading cause of cancer-related deaths in women. Triple negative breast cancer (TNBC) is an aggressive subtype that affects 10–25% mostly African American women. TNBC has the poorest prognosis of all subtypes with rapid progression leading to mortality in younger patients. So far, there is no targeted treatment for TNBC. To that end, here we show that c-Abl is one of several tyrosine kinases that phosphorylate and activate geminin’s ability to promote TNBC. Analysis of >800 breast tumor samples showed that geminin is overexpressed in ∼50% of all tumors. Although c-Abl is overexpressed in ∼90% of all tumors, it is only nuclear in geminin overexpressing tumors. In geminin-negative tumors, c-Abl is only cytoplasmic. Inhibiting c-Abl expression or activity (using imatinib or nilotinib) prevented geminin Y150 phosphorylation, inactivated the protein, and most importantly converted overexpressed geminin from an oncogene to an apoptosis inducer. In pre-clinical orthotopic breast tumor models, geminin-overexpressing cells developed aneuploid and invasive tumors, which were suppressed when c-Abl expression was blocked. Moreover, established geminin overexpressing orthotopic tumors regressed when treated with imatinib or nilotinib. Our studies support imatinib/nilotonib as a novel treatment option for patients with aggressive breast cancer (including a subset of TNBCs)-overexpressing geminin and nuclear c-Abl.

MTA1‐activated Epi‐microRNA‐22 regulates E‐cadherin and prostate cancer invasiveness

We have previously shown that metastasis‐associated protein 1 (MTA1), a chromatin remodeler, plays an important role in prostate cancer invasiveness, likely through regulation of epithelial‐to‐mesenchymal transition. Here, we identified miR‐22 as an epigenetic‐microRNA (Epi‐miR) directly induced by MTA1 and predicted to target E‐cadherin. Loss‐of‐function and overexpression studies of MTA1 reinforced its regulatory role in miR‐22 expression. MiR‐22 directly targets the 3′‐untranslated region of E‐cadherin, and ectopic overexpression of miR‐22 diminishes E‐cadherin expression. Overexpression of miR‐22 in prostate cancer cells promotes cell invasiveness and migration. Meta‐analysis of patient tumor samples indicates a positive correlation between MTA1 and miR‐22, supporting their inhibitory effect on E‐cadherin expression. Our findings implicate the MTA1/Epi‐miR‐22/E‐cadherin axis as a new epigenetic signaling pathway that promotes tumor invasion in prostate cancer.

Targeting MTA1/HIF-1α signaling by pterostilbene in combination with histone deacetylase inhibitor attenuates prostate cancer progression

The metastasis‐associated protein 1(MTA1)/histone deacetylase (HDAC) unit is a cancer progression‐related epigenetic regulator, which is overexpressed in hormone‐refractory and metastatic prostate cancer (PCa). In our previous studies, we found a significantly increased MTA1 expression in a prostate‐specific Pten‐null mouse model. We also demonstrated that stilbenes, namely resveratrol and pterostilbene (Pter), affect MTA1/HDAC signaling, including deacetylation of tumor suppressors p53 and PTEN. In this study, we examined whether inhibition of MTA1/HDAC using combination of Pter and a clinically approved HDAC inhibitor, SAHA (suberoylanilide hydroxamic acid, vorinostat), which also downregulates MTA1, could block prostate tumor progression in vivo. We generated and utilized a luciferase reporter in a prostate‐specific Pten‐null mouse model (Pb‐Cre+; Ptenf/f; Rosa26Luc/+) to evaluate the anticancer efficacy of Pter/SAHA combinatorial approach. Our data showed that Pter sensitized tumor cells to SAHA treatment resulting in inhibiting tumor growth and additional decline of tumor progression. These effects were dependent on the reduction of MTA1‐associated proangiogenic factors HIF‐1α, VEGF, and IL‐1β leading to decreased angiogenesis. In addition, treatment of PCa cell lines in vitro with combined Pter and low dose SAHA resulted in more potent inhibition of MTA1/HIF‐1α than by high dose SAHA alone. Our study provides preclinical evidence that Pter/SAHA combination treatment inhibits MTA1/HIF‐1α tumor‐promoting signaling in PCa. The beneficial outcome of combinatorial strategy using a natural agent and an approved drug for higher efficacy and less toxicity supports further development of MTA1‐targeted therapies in PCa.

Pathology and Genomics in Gestational Trophoblastic Neoplasia

Gestational trophoblastic diseases (GTDs) originate from placental tissue and are rare tumors with a current cure rate of greater than 90% with the right diagnosis and clinical management. GTDs are generally divided into two categories: (1) hydatidiform moles, presenting abnormal villous proliferation with chromosomal aberrations, and (2) rare gestational trophoblastic neoplasms (GTNs) including choriocarcinoma, placental site trophoblastic tumor, and epithelioid trophoblastic tumor. Persistent gestational trophoblastic disease/tumor most commonly occurs following molar pregnancy; however, it may follow any GTD.

Abstract 4973: The role of cholesteryl esters in progression and racial disparity of prostate cancer

Background Prostate cancer (PCa) is featured by uncertainty of future outcomes at the time of diagnosis, development of castration resistant prostate cancer (CRPC), and racial disparity in morbidity and mortality, which is especially prominent between African American (AA) and Caucasian American (CA). This study is aimed to investigate whether accumulation of cholesteryl esters (CE) in PCa is a key biological process that determines progression potential, and serves as a common mechanism underlying these features. Methods The methods used in this study include: 1) ESI/MS-MS on 47 fresh-frozen prostatic tissues for global lipid profiling; 2) Real-time PCR on 16 fresh-frozen prostatic tissues for the expression level of genes related to the pathogenesis of prostate cancer and metabolism of cholesteryl esters; and 3) immunohistochemistry on 165 formalin-fixed and paraffin embedded prostatic tissues for the expression level of ACAT1 and LAL. Results We found that 1) prostatic concentration of total lipids was significantly higher in PCa than benign prostatic tissues (BPT), with a PCa to BPT (P/B) ratio of 2.3, p = 0.013; 2) when total lipids were stratified into groups of neutral lipids and polar lipids, only neutral lipids are significantly higher in PCa than BPT (P/B ratio: 3.1, p = 0.02); 3) after neutral lipids were further stratified into class of triglycerides, diglycerides, free fatty acids (FFA) and CEs, total CEs are most sensitive in differentiation of PCa from BPT (P/B ratio: 5.8, p = 0.025); 4) 17 out of 31 prostatic individual CE species are significantly higher in PCa than in BPT all the studied populations; and 5) intriguingly, 14 of these 17 prostatic CE individual species remain significantly different between PCa and BPT in AA population, whereas only 3 of them remain significantly different between PCa and BPT in the CA population. The real-time PCR results indicated that the expression levels of genes Pten, LIPA, and ABCA1 are obviously lower in high grade than in low grade PCa (not obviously for ACAT1). The expression levels of proteins ACAT1 and LAL (LIPA gene product) are reversely correlated with the progression of PCa: in prostate cancer, ACAT1 is highly expressed, but LAL is not expressed. In contrary, in benign ACAT1 is not expressed, but LAL is highly expressed. Such a reverse correlation is especially prominent in AA than CA population. Conclusion and Significance Accumulation of cholesterol esters correlates with the progression and racial disparity of PCa. Down regulation of LIPA gene and its product LAL could play major role in CE accumulation. Thus prostatic CE and its individual species could serve as racially specific biomarkers in diagnosis and in differentiation of indolent from aggressive cases of PCa, and LAL could be potential therapeutic targets for treatment of advanced prostate cancer, including castration resistant prostate cancer. Citation Format: Xinchun Zhou, Timera Brown, Janice Lage, Jinghe Mao. The role of cholesteryl esters in progression and racial disparity of prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4973.

Abstract 5003: Differential expression of lysophosphatidic acid receptors (LPARs) between benign and malignant tissues in humans

Background: Lysophosphatidic acid receptors (LPARs) are important to normal cellular functions and implicated in the pathogenesis and survival of various cancers recently. Previous studies were mostly focused on one LPAR using experiments on animal models and cell cultures. This study was executed to investigate systemic distribution of LPAR1, LPAR2, and LPAR3 in human organs/tissues and to compare the expression levels of these receptors between benign and malignant changes in major human organs/tissues. Method: Tissue Microarrays TMAs) were constructed with 384 formalin-fixed and paraffin-embedded benign and malignant specimens from 33 different human organs/tissues. Immunohistochemistry (IHC) for each LPAR was performed on the TMA slides. In addition to observing the subcellular localization of each LPAR, the expression level of each LPAR at each subcellular localization was recorded as final IHC score, calculated by IHC intensity score (0 to3) multiplied with IHC area score (0 to 3). A 2-sample t test was used to statistical analysis. Results: All three LPARs were widely distributed throughout human organs/tissues. All LPARs were expressed higher in nuclei than in cytoplasm. Nuclear expressed LPARs were all higher in overall malignant changes than in overall benign changes, which is especially true for nuclear expressed LPAR1 (LPAR1N): the IHC score for LPAR1N was 6.38 in overall malignant changes and 5.84 in overall benign changes (p = 0.016). The expression level of LPAR1 in nuclei or in cytoplasm is significantly (p 0.05) different between benign and malignant changes in eight organs/tissues. The number of organs/tissues with such difference was five for LPAR2 and six for LPAR3. Hepatocellular carcinoma (HCC) had significantly higher expression level of LPAR1N (p = 0.000000001), LPAR2N (p = 0.00002) and LPAR3N (p = 0.00001) as compared with benign hepatic changes. The malignancies showed obviously alterations in expression level of two LPARs in three organs/tissues and one LPAR in eight organs/tissues. The malignancies in the rest organs/tissues, including breast and prostate showed no obvious difference between benign and malignant changes. Conclusions: This study is first to investigate systemic distribution of the LPAR1, LPAR2 and LPAR3 in benign and malignant changes throughout human organs/tissues. The results indicate that each LPAR has a unique expression pattern in a spectrum of organs/tissues. Differentially expressed LPARs between benign and malignant changes in different organs/tissues will help to understand the involvement of LPARs in the pathogenesis of cancer, and to find therapeutic targets in treatment of cancer. Citation Format: Andrew Bingham, Jinghe Mao, Chunyi Wang, Janice Lage, Xinchun Zhou. Differential expression of lysophosphatidic acid receptors (LPARs) between benign and malignant tissues in humans. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5003. doi:10.1158/1538-7445.AM2015-5003

Abstract 3199: Pterostilbene supresses prostate cancer progression in transgenic mice model

Metastasis-associated protein 1 (MTA1) is a cancer progression-related epigenetic modifier whose overexpression is associated with aggressive prostate cancer (PCa), disease recurrence and metastasis in men. We previously found that MTA1 expression is significantly increased in Pten-null murine cells, and that resveratrol and its analogues inhibit MTA1 expression. In the current study, we used the murine Pten knockout PCa model to evaluate the effect of pterostilbene (PTER) on MTA1-mediated events in PCa progression. Pterostilbene is a potent natural analogue of resveratrol with improved pharmacokinetic properties and superior pharmacological potency. The Pten conditional knockout mice that mimic human disease and provide fast stage-defined development of PCa were fed, starting at 3 weeks of age, with phytoestrogen-free diet AIN 76A and injected daily (i.p.) either with 10% DMSO (control, untreated) or with 10 mg/kg bw PTER until 6, 10, 15 and 25 weeks of age. At necropsy, mouse prostates were formalin-fixed and paraffin-embedded for histological and immunohistochemical analysis. For protein analysis, mouse prostate dissection was performed to distinguish anterior prostate (AP) and dorso-latero-ventral (DLV) lobes. Our results showed that the PTER-treated group had a lower tumor grade and higher incidence of prostate intraepithelial neoplasia (PIN) at the expense of a substantial decrease in adenocarcinoma incidence. Prostate tissues from the PTER-treated group presented a decrese in Ki67 proliferation marker-positive cells and an increase in apoptosis. As potential mechanisms of PTER efficacy, 50-75% decrease in MTA1 and pAkt levels were observed at different time points. In addition, PTER inhibited androgen receptor (AR) expression, especially in the AP. Taken together, our findings suggest that PTER blocks PCa growth and progression at PIN stage in Pten conditional knockout mice model through inhibition of MTA1-Akt axis. Citation Format: Liangfen Zhang, Swati Dhar, Agnes M. Rimando, Avinash Kumar, Janice Lage, Jack R. Lewin, Xu Zhang, Anait S. Levenson. Pterostilbene supresses prostate cancer progression in transgenic mice model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3199. doi:10.1158/1538-7445.AM2014-3199