Paula Sotomayor

Oct4A is expressed by a subpopulation of prostate neuroendocrine cells.

Cancer stem cells are defined by their self‐renewal and multi‐potential capabilities and are hypothesized to be the source of primary and recurrent cancers. The stem cell properties of self‐renewal and pluripotency in embryonic stem cells and germ cells are regulated by Oct4A, a splice variant of the POU5F1 (Oct3/4) gene, while the function of the alternative splice variant, Oct4B, is unknown. Rare cells that express Oct4 were identified in several somatic cancers, however, the differential contributions of the Oct4A and Oct4B variants were not determined.

Class I histone deacetylase inhibitor entinostat suppresses regulatory T cells and enhances immunotherapies in renal and prostate cancer models.

Background Immunosuppressive factors such as regulatory T cells (Tregs) limit the efficacy of immunotherapies. Histone deacetylase (HDAC) inhibitors have been reported to have antitumor activity in different malignancies and immunomodulatory effects. Herein, we report the Tregs-targeting and immune-promoting effect of a class I specific HDAC inhibitor, entinostat, in combination with either IL-2 in a murine renal cell carcinoma (RENCA) model or a survivin-based vaccine therapy (SurVaxM) in a castration resistant prostate cancer (CR Myc-CaP) model. Methods and Results RENCA or CR Myc-CaP tumors were implanted orthotopically or subcutaneously, respectively. Inoculated mice were randomized into four treatment groups: vehicle, entinostat, cytokine or vaccine, and combination. Tregs in the blood were assessed by FACS analysis. Real time quantitative PCR and Western blot analysis of isolated T cell subpopulations from spleen were performed to determine Foxp3 gene and protein expression. The suppressive function of Tregs was tested by T cell proliferation assay. Low dose (5 mg/kg) entinostat reduced Foxp3 levels in Tregs and this was associated with enhanced tumor growth inhibition in combination with either IL-2 or a SurVaxM vaccine. Entinostat down-regulated Foxp3 expression transcriptionally and blocked Tregs suppressive function without affecting T effector cells (Teffs). In vitro low dose entinostat (0.5 µM) induced STAT3 acetylation and a specific inhibitor of STAT3 partially rescued entinostat-induced down-regulation of Foxp3, suggesting that STAT3 signaling is involved in Foxp3 down-regulation by entinostat. Conclusions These results demonstrate a novel immunomodulatory effect of class I HDAC inhibition and provide a rationale for the clinical testing of entinostat to enhance cancer immunotherapy.

Mechanistic Insights and Functional Determinants of the Transport Cycle of the Ascorbic Acid Transporter SVCT2 ACTIVATION BY SODIUM AND ABSOLUTE DEPENDENCE ON BIVALENT CATIONS

We characterized the human Na+-ascorbic acid transporter SVCT2 and developed a basic model for the transport cycle that challenges the current view that it functions as a Na+-dependent transporter. The properties of SVCT2 are modulated by Ca2+/Mg2+ and a reciprocal functional interaction between Na+ and ascorbic acid that defines the substrate binding order and the transport stoichiometry. Na+ increased the ascorbic acid transport rate in a cooperative manner, decreasing the transport Km without affecting the Vmax, thus converting a low affinity form of the transporter into a high affinity transporter. Inversely, ascorbic acid affected in a bimodal and concentration-dependent manner the Na+ cooperativity, with absence of cooperativity at low and high ascorbic acid concentrations. Our data are consistent with a transport cycle characterized by a Na+:ascorbic acid stoichiometry of 2:1 and a substrate binding order of the type Na+:ascorbic acid:Na+. However, SVCT2 is not electrogenic. SVCT2 showed an absolute requirement for Ca2+/Mg2+ for function, with both cations switching the transporter from an inactive into an active conformation by increasing the transport Vmax without affecting the transport Km or the Na+ cooperativity. Our data indicate that SVCT2 may switch between a number of states with characteristic properties, including an inactive conformation in the absence of Ca2+/Mg2+. At least three active states can be envisioned, including a low affinity conformation at Na+ concentrations below 20 mm and two high affinity conformations at elevated Na+ concentrations whose Na+ cooperativity is modulated by ascorbic acid. Thus, SVCT2 is a Ca2+/Mg2+-dependent transporter.

Vitamin C Is an Essential Antioxidant That Enhances Survival of Oxidatively Stressed Human Vascular Endothelial Cells in the Presence of a Vast Molar Excess of Glutathione

Cellular glutathione levels may exceed vitamin C levels by 10-fold, generating the question about the real antioxidant role that low intracellular concentrations of vitamin C can play in the presence of a vast molar excess of glutathione. We characterized the metabolism of vitamin C and its relationship with glutathione in primary cultures of human endothelial cells oxidatively challenged by treatment with hydrogen peroxide or with activated cells undergoing the respiratory burst, and analyzed the manner in which vitamin C interacts with glutathione to increase the antioxidant capacity of cells. Our data indicate that: (i) endothelial cells express transporters for reduced and oxidized vitamin C and accumulate ascorbic acid with participation of glutathione-dependent dehydroascorbic acid reductases, (ii) although increased intracellular levels of vitamin C or glutathione caused augmented resistance to oxidative stress, 10-times more glutathione than vitamin C was required, (iii) full antioxidant protection required the simultaneous presence of intracellular and extracellular vitamin C at concentrations normally found in vivo, and (iv) intracellular vitamin C cooperated in enhancing glutathione recovery after oxidative challenge thus providing cells with enhanced survival potential, while extracellular vitamin C was recycled through a mechanism involving the simultaneous neutralization of oxidant species. Therefore, in endothelial cells under oxidative challenge, vitamin C functions as an essential cellular antioxidant even in the presence of a vast molar excess of glutathione.

Rb-dependent cellular senescence, multinucleation and susceptibility to oncogenic transformation through PKC scaffolding by SSeCKS/AKAP12

A subset of AKAPs (A Kinase Anchoring Proteins) regulate signaling and cytoskeletal pathways through the spaciotemporal scaffolding of multiple protein kinases (PK) such as PKC and PKA, and associations with the plasma membrane and the actin-based cytoskeleton. SSeCKS/Gravin/Akap12 expression is severely downregulated in many advanced cancers and exhibits tumor- and metastasis-suppressing activity. akap12-null (KO) mice develop prostatic hyperplasia with focal dysplasia, but the precise mechanism how Akap12 prevents oncogenic progression remains unclear. Here, we show that KO mouse embryonic fibroblasts (MEF) exhibit premature senescence marked by polyploidy and multinucleation, and by increased susceptibility to oncogenic transformation. Although p53 and Rb pathways are activated in the absence of Akap12, senescence is dependent on Rb. Senescence is driven by the activation of PKCα, which induces p16Ink4a/Rb through a MEK-dependent downregulation of Id1, and PKCδ, which downregulates Lats1/Warts, a mitotic exit network kinase required for cytokinesis. Our data strongly suggest that Akap12 controls Rb-mediated cell aging and oncogenic progression by directly scaffolding and attenuating PKCα/δ.

ABCG2-mediated DyeCycle Violet efflux defined side population in benign and malignant prostate

The efflux of Hoechst 33342 by ATP-binding cassette protein G2 (ABCG2) membrane pump allows reproducible identification of a subpopulation of cells by flow cytometric analysis termed the “side population” (SP). The SP identified by constitutive Hoechst efflux contains the stem/progenitor cell population from bone marrow and many solid organs, including prostate. DyeCycle Violet (DCV) is a cell membrane permeable, fluorescent vital dye that intercalates into DNA and is a substrate for ABCG2-mediated efflux. Therefore, DCV was evaluated in this study as a tool for identification of the SP from prostate cancer cell lines and from freshly harvested human prostate tissue. SPs that demonstrated ABCG2-mediated efflux of DCV were identified in the human prostate cancer cell lines CWR-R1, DU-145, and RWPE-1, but not in the BPH-1, LAPC-4, or PC-3 cell lines. Additionally, a SP was identified in enzymatically disaggregated prostate tumors from Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) human benign prostate tissue, and human prostate cancer tissue. The causal role of ABCG2-mediated efflux of DCV in the identification of the SP was confirmed by loss of the SP by incubation with the specific inhibitor of ABCG2, Fumitremorgin C. Expression of ABCG2 in the SP cells was confirmed by qRT-PCR and immunofluorescence analysis. Consequently, DCV represents an important new tool for isolation of viable candidate stem cells/cancer stem cells as a SP from cultured prostate cell lines, and prostate tissue specimens, without the requirement for instrumentation with ultra-violet excitation capability and minimizing the risk of damage to DNA in the sorted population.

Cellular distribution of Glut-1 and Glut-5 in benign and malignant human prostate tissue†

Over‐expression of hexose transporters (Gluts), specifically Glut‐1, is a common event in human malignancies. In prostate cancer (CaP), however, expression of Gluts has been characterized poorly. In this study, expression and distribution of Glut‐1 and Glut‐5 proteins were characterized using immunohistochemistry in 76 specimens of benign prostate, 10 specimens of high‐grade intraepithelial neoplasia (HGPIN), and 28 specimens of CaP. In addition, mRNA expression of Glut‐2, Glut‐7, Glut‐9, and Glut‐11 was analyzed in a set of five specimens of benign prostate and CaP. In benign prostate, Glut‐1 localized to the basal cells and to the basolateral membrane of secretory/luminal epithelial cells. Glut‐5, however, localized to the apical membrane of secretory/luminal epithelial cells. In HGPIN, Glut‐1 was immunohistochemically undetectable. Glut‐5, however, localized to the apical membrane of the neoplastic epithelial cells. In CaP, Glut‐1 and Glut‐5, were immunohistochemically undetectable. However, over‐expression of GLUT1 was observed in some specimens of highly proliferative intraductal CaP. Glut‐7, Glut‐9, and Glut‐11 mRNAs were detected in benign prostate and CaP, however, only Glut‐11 mRNA was consistently up‐regulated in CaP compared to benign prostate. Low levels of expression of Glut‐1 protein in the majority of CaP could explain, at least in part, the limited clinical applicability of positron emission tomography using 2‐[18F]‐fluoro‐2‐deoxy‐D‐glucose for imaging CaP. Moreover, expression of Glut‐5 in HGPIN suggested that fructose could be utilized as potential metabolic substrate in HGPIN. Understanding the molecular mechanisms involved in regulation/dysregulation of Gluts in CaP could provide insight in the understanding of hexose metabolism in CaP. J. Cell. Biochem. 113: 553–562, 2012.

Induction of cell cycle arrest and DNA damage by the HDAC inhibitor panobinostat (LBH589) and the lipid peroxidation end product 4-hydroxynonenal in prostate cancer cells

Histone deacetylase inhibitors (HDACIs) are promising antineoplastic agents for the treatment of cancer. Here we report that the lipid peroxidation end product 4-hydroxynonenal (HNE) significantly potentiates the anti-tumor effects of the HDAC inhibitor panobinostat (LBH589) in the PC3 prostate cancer cell model. Panobinostat and HNE inhibited proliferation of PC3 cells and the combination of the two agents resulted in a significant combined effect. Cell cycle analysis revealed that both single agents and, to a greater extent, their combined treatment induced G2/M arrest, but cell death occurred in the combined treatment only. Furthermore, HNE and, to a greater extent, the combined treatment induced dephosphorylation of Cdc2 leading to progression into mitosis as confirmed by α-tubulin/DAPI staining and phospho-histone H3 (Ser10) analysis. To evaluate possible induction of DNA damage we utilized the marker phosphorylated histone H2A.X. Results showed that the combination of panobinostat and HNE induced significant DNA damage concomitant with the mitotic arrest. Then, by using androgen receptor (AR)-expressing PC3 cells we observed that the responsiveness to HNE and panobinostat was independent of the expression of functional AR. Taken together, our data suggest that HNE potentiates the antitumoral effect of the HDACI panobinostat in prostate cancer cells.

Characterization of iNOS + Neutrophil-like ring cell in tumor-bearing mice

BackgroundMyeloid-derived Suppressor Cells (MDSC) have been identified as tumor-induced immature myeloid cells (IMC) with potent immune suppressive activity in cancer. Whereas strict phenotypic classification of MDSC has been challenging due to the highly heterogeneous nature of cell surface marker expression, use of functional markers such as Arginase and inducible nitric oxide synthase (iNOS) may represent a better categorization strategy. In this study we investigated whether iNOS could be utilized as a specific marker for the identification of a more informative homogenous MDSC subset.MethodsSingle-cell suspensions from tumors and other organs were prepared essentially by enzymatic digestion. Flow cytometric analysis was performed on a four-color flow cytometer. Morphology, intracellular structure and localization of iNOS+ ring cells in the tumor were determined by cytospin analysis, immunofluorescence microscopy and immunohistochemistry, respectively. For functional analysis, iNOS+ ring subset were sorted and tested in vitro cell culture experiments. Pharmacologic inhibition of iNOS was performed both in vivo and in vitro.ResultsThe results showed that intracellular iNOS staining distinguished a granular iNOS+ SSChi CD11b+ Gr-1dim F4/80+ subset with ring-shaped nuclei (ring cells) among the CD11b+ Gr-1+ cell populations found in tumors. The intensity of the ring cell infiltrate correlated with tumor size and these cells constituted the second major tumor-infiltrating leukocyte subset found in established tumors. Although phenotypic analysis demonstrated that ring cells shared characteristics with tumor-associated macrophages (TAM), morphological analysis revealed a neutrophil-like appearance as detected by cytospin and immunofluorescence microscopy analysis. The presence of distinct iNOS filled granule-like structures located next to the cell membrane suggested that iNOS was stored in pre-formed vesicles and available for rapid release upon activation. Tumor biopsies showed large areas with infiltrating ring cells primarily surrounding necrotic areas. Importantly, these cells significantly impaired CD8+ T-cell proliferation and induced apoptotic death. The intratumoral accumulation and suppressive activity of ring cells could be blocked through pharmacologic inhibition of iNOS, demonstrating the critical role of this enzyme in mediating both the differentiation and the activity of these cells.ConclusionsIn this study, iNOS expression was linked to a homogeneous subset; ring cells with a particular phenotype and immune suppressive function, in a common and well-established murine tumor model; 4T-1. Since the absence of a Gr-1 homolog in humans has made the identification of MDSC much more challenging, use of iNOS as a functional marker of MDSC may also have clinical importance.

Sunitinib Dose Escalation Overcomes Transient Resistance in Clear Cell Renal Cell Carcinoma and Is Associated with Epigenetic Modifications

Sunitinib is considered a first-line therapeutic option for patients with advanced clear cell renal cell carcinoma (ccRCC). Despite sunitinibs clinical efficacy, patients eventually develop drug resistance and disease progression. Herein, we tested the hypothesis whether initial sunitinib resistance may be transient and could be overcome by dose increase. In selected patients initially treated with 50 mg sunitinib and presenting with minimal toxicities, sunitinib dose was escalated to 62.5 mg and/or 75 mg at the time of tumor progression. Mice bearing two different patient-derived ccRCC xenografts (PDX) were treated 5 days per week with a dose-escalation schema (40–60–80 mg/kg sunitinib). Tumor tissues were collected before dose increments for immunohistochemistry analyses and drug levels. Selected intrapatient sunitinib dose escalation was safe and several patients had added progression-free survival. In parallel, our preclinical results showed that PDXs, although initially responsive to sunitinib at 40 mg/kg, eventually developed resistance. When the dose was incrementally increased, again we observed tumor response to sunitinib. A resistant phenotype was associated with transient increase of tumor vasculature despite intratumor sunitinib accumulation at higher dose. In addition, we observed associated changes in the expression of the methyltransferase EZH2 and histone marks at the time of resistance. Furthermore, specific EZH2 inhibition resulted in increased in vitro antitumor effect of sunitinib. Overall, our results suggest that initial sunitinib-induced resistance may be overcome, in part, by increasing the dose, and highlight the potential role of epigenetic changes associated with sunitinib resistance that can represent new targets for therapeutic intervention. Mol Cancer Ther; 14(2); 513–22. ©2014 AACR.