Ingrid Espinoza

Deadly crosstalk: Notch signaling at the intersection of EMT and cancer stem cells

Notch signaling is an evolutionarily conserved pathway involved in cell fate control during development, stem cell self-renewal and postnatal tissue differentiation. Roles for Notch in carcinogenesis, in the biology of cancer stem cells, tumor angiogenesis and epithelial-to-mesenchymal transition (EMT) have been reported. This mini-review describes the role of Notch signaling deregulation in EMT and tumor aggressiveness. We describe how accumulated evidence suggests that Notch inhibition is an attractive strategy for the treatment of several cancers, at least in part because of its potential to reverse or prevent EMT.

Notch signaling: targeting cancer stem cells and epithelial-to-mesenchymal transition

Notch signaling is an evolutionarily conserved pathway involved in cell fate control during development, stem cell self-renewal, and postnatal tissue differentiation. Roles for Notch in carcinogenesis, the biology of cancer stem cells, tumor angiogenesis, and epithelial-to-mesenchymal transition (EMT) have been reported. This review describes the role of Notch in the “stemness” program in cancer cells and in metastases, together with a brief update on the Notch inhibitors currently under investigation in oncology. These agents may be useful in targeting cancer stem cells and to reverse the EMT process.

Elevated Expression of Hepatoma Up-Regulated Protein Inhibits γ-Irradiation-Induced Apoptosis of Prostate Cancer Cells.

Despite progression in diagnosis and treatment, prostate cancer (PCa) still represents the main cause of cancer‐related mortality and morbidity in men. Although radiation therapy offers clinical benefit over other therapeutic modalities, the success of this therapeutic modality is commonly hampered by the resistance of advanced tumors. So far, the mechanisms governing tumor resistance to radiotherapy are not discussed in detail. Here, we demonstrate for the first time that the resistance of PCa to radiation therapy is attributed to elevated expression of Hepatoma Up‐Regulated Protein (HURP). In PCa cells, the induction of HURP expression suppresses γ‐irradiation‐induced apoptosis. γ‐irradiation‐induced apoptosis of PCa cells is associated with expression of E2F1, p53, p21 proteins together with the phosphorylation of apoptosis signal‐regulating kinase1 (ASK1), c‐jun‐N‐terminal kinase (JNK) and Ataxia‐telangiectasia mutated (ATM) and histone family member X (H2AX). Whereas, the induction of HURP expression is able to suppress γ‐irradiation‐induced effects on E2F1, p53, p21, ATM, ASK1, JNK and ATM, and H2AX. Also, inhibition of γ‐irradiation‐induced‐ cytochrome c release, cleavage of caspase‐9, caspase‐3, PARP, and reactive oxygen species (ROS) were noted in PCa cells induced for HURP expression. The observed radio‐resistance of PCa is thought to be the consequence of HURP‐mediated destabilization of p53 and ATM proteins that are essential for the modulation of γ‐irradiation‐induced apoptosis. Thus, based on our findings, PCa resistance to radiation therapy results from the deregulation of ASK1/ JNK; ATM/ H2AX; ATM/p53 and checkpoint kinase 2 (Chk2)/ E2F‐1 in response to the elevated expression of HURP. J. Cell. Biochem. 117: 1308–1318, 2016.

Cross-sectional association of exercise, strengthening activities, and cardiorespiratory fitness on generalized anxiety, panic and depressive symptoms

ABSTRACT Objective: Limited research has evaluated the individual and combined associations of physical activity (PA), cardiorespiratory fitness (CRF) and muscle strengthening activities (MSA) on generalized anxiety, panic and depressive symptoms. We evaluated this topic in a representative sample of young (20–39 years) adults, with considerations by sex. Methods: Data from the 1999–2004 National Health and Nutrition Examination Survey (N = 2088) were used. Generalized anxiety, panic and depressive symptoms were assessed via self-report as well as using the Generalized Anxiety Disorder, Panic Disorder, and Depressive Disorders modules of the automated version of the World Health Organization Composite International Diagnostic Interview (CIDI-Auto 2.1). PA and MSA were assessed via validated self-report questionnaires and CRF was determined via a submaximal treadmill-based test. An index variable was created summing the number (range = 0–3) of these parameters for each participant. For example, those meeting PA guidelines, MSA guidelines and having moderate-to-high CRF were classified as having an index score of 3. Results: MSA was not independently associated with generalized anxiety, panic and depressive symptoms, but those with higher levels of PA and CRF had a reduced odds of these symptoms (ranging from 40 to 46% reduced odds). Compared to those with an index score of 0, those with an index score of 1, 2, and 3, respectively, had a 39%, 54% and 71% reduced odds of having generalized anxiety, panic and depressive symptoms. Results were consistent across both sexes. Conclusion: PA and CRF, but not MSA, were independently associated with generalized anxiety, panic and depressive symptoms. There was evidence of an additive association between PA, CRF, and MSA on these symptoms.

Hypoxia on the Expression of Hepatoma Upregulated Protein in Prostate Cancer Cells

Hepatoma upregulated protein (HURP) is a multifunctional protein with clinical promise. This protein has been demonstrated to be a predictive marker for the outcome in high-risk prostate cancer (PCa) patients, besides being a resistance factor in PCa. Although changes in oxygen tension (pO2) are associated with PCa aggressiveness, the role of hypoxia in the regulation of tumor progression genes such as HURP has not yet been described. We hypothesized that pO2 alteration is involved in the regulation of HURP expression in PCa cells. In the present study, PCa cells were incubated at 2% O2 (hypoxia) and 20% O2 (normoxia) conditions. Hypoxia reduced cell growth rate of PCa cells, when compared to the growth rate of cells cultured under normoxia (p < 0.05). The decrease in cell viability was accompanied by fivefold (p < 0.05) elevated rate of vascular endothelial growth factor (VEGF) release. The expression of VEGF and the hypoxia-inducible metabolic enzyme carbonic anhydrase 9 were elevated maximally nearly 61-fold and 200-fold, respectively (p < 0.05). Noted in two cell lines (LNCaP and C4-2B) and independent of the oxygen levels, HURP expression assessed at both mRNA and protein levels was reduced. However, the decrease was more pronounced in cells cultured under hypoxia (p < 0.05). Interestingly, the analysis of patients’ specimens by Western blot revealed a marked increase of HURP protein (fivefold), when compared to control (cystoprostatectomy) tissue (p < 0.05). Immunohistochemistry analysis showed an increase in the immunostaining intensity of HURP and the hypoxia-sensitive molecules, hypoxia-inducible factor 1-alpha (HIF-1α), VEGF, and heat-shock protein 60 (HSP60) in association with tumor grade. The data also suggested a redistribution of subcellular localization for HURP and HIF-1α from the nucleus to the cytoplasmic compartment in relation to increasing tumor grade. Analysis of HURP Promoter for HIF-1-binding sites revealed presence of four putative HIF binding sites on the promoter of DLGAP5/HURP gene in the non-translated region upstream from the start codon, suggesting association between HIF-1α and the regulation of HURP protein. Taken together, our findings suggest a modulatory role of hypoxia on the expression of HURP. Additionally our results provide basis for utilization of tumor-associated molecules as predictors of aggressive PCa.

Genetic Mutations in B-Acute Lymphoblastic Leukemia Among African American and European American Children

Background: The survival of patients with B‐acute lymphoblastic leukemia (B‐ALL) is significantly lower in African American (AA) children compared with European American children (EA). Here, we present a whole exome sequencing (WES) study showing race‐specific genetic variations that may play a role on the disparate outcomes among AA and EA children with B‐ALL. Patients and Methods: Five AA and 15 EA patients ranging in age from 1 to 18 years were enrolled. The median blast percentage was 94.8% (range, 64.5%‐99.9%). Frozen bone marrow aspirate was used to extract DNA, and WES was performed, focusing on race and B‐ALL‐specific germline mutations. Results: Most genetic variants (n = 339) were shared between AA and EA children. Some genetic aberrations were only uniquely identified in AA (n = 58) and others in EA (n = 52) In AA, the genetic aberrations clustered in canonical pathways related to telomerase signaling and cancer signaling. In EA, the unique genetic aberration clustered in pathways related to stem cell pluripotency and hereditary cancer. Conclusions: Our study revealed aberrant genetic aberrations in signaling networks that may contribute to race‐specific aspects of leukemogenesis. Our results suggest the value of WES as a tool for development of individual gene signatures and gene scores for AA and EA children afflicted by B‐ALL. These findings may ultimately impact disease management and contribute to the elimination of disparate outcomes in AA children with B‐ALL.

Abstract 4918: Emerging prognostic biomarkers in colorectal cancer: HURP and ZEB1

The current diagnosis of colorectal cancer (CRC) is based on tumor-node-metastasis staging. This classification system has weaknesses due to different genetic and epigenetic backgrounds. Biological markers would improve early detection and guide clinicians in subsequent treatment decisions. Stem-like molecules have the potential to identify patients at high risk of developing aggressive carcinomas. Herein, Hepatoma Up-Regulate Protein (HURP) and Zinc finger E-box binding homeobox 1 (ZEB1) are assessed as potential prognostic biomarkers in CRC. Tumors (N = 21) obtained from colectomies and 5 μm formalin-fixed paraffin-embedded blocks were sectioned and immunostained for HURP and ZEB1. For certain cases (N = 8), flash frozen tissues from tumor and adjacent normal colonic tissue were used to extract total RNA, synthesize cDNA, and perform RT-PCR. Presence of tumor cells in analyzed tissue sections was assessed by a pathologist. HURP mRNA expression was 4-fold higher (p = 0.0005) in tumor tissue relative to adjacent normal tissue. Furthermore, an increase in HURP expression was evident in all tumor samples relative to their respective normal tissue control with an average 3-fold increase (p = 0.001). Immunohistochemical analysis (IHC) of HURP revealed expression in all analyzed specimens. Although the stroma was weakly stained for HURP in some cases, all tumors were immunostained for this protein (average intensity score = ++, p Citation Format: Logan Fair, Ingrid Espinoza, Xu Zhang, Abdelouahid Elkhattouti, Tangeng Ma, Joy King, Elizabeth Tarsi, Richard Whitlock, Vijay Kannuthurai, Ryan Jimenez, Tara Craft, Mary Graichen, Sharon Lobert, Roy Duhe, Charulochana Subramony, Christopher Lahr, Christian R. Gomez. Emerging prognostic biomarkers in colorectal cancer: HURP and ZEB1. [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 4918.

Abstract B03: HURP and ZEB1: Novel prognostic biomarkers in colorectal carcinomas

The current clinicopathologic diagnosis of colorectal cancer (CRC) is based on tumor-node-metastasis staging. This classification system has weaknesses due to the different genetic and epigenetic backgrounds not currently incorporated into CRC staging which can ultimately lead to treatment failure. Biological markers would improve early detection and guide clinicians in subsequent treatment decisions. Recently, we and others have identified “stem-like” molecules with the potential to identify patients at high risk of developing aggressive carcinomas. Herein, Hepatoma Up-Regulate Protein (HURP) and Zinc finger E-box binding homeobox 1 (ZEB1) are assessed as potential prognostic biomarkers in CRC. Tumors (N=21) obtained from colectomies and 5 µm formalin-fixed paraffin-embedded blocks were sectioned and immunostained for HURP and ZEB1. For certain cases (N=8), flash frozen tissues from tumor and adjacent normal colonic tissue were used to extract total RNA, synthesize cDNA, and perform RT-PCR. Presence of tumor cells in analyzed tissue sections was assessed by a pathologist. HURP mRNA expression was 4-fold higher ( p =0.0005) in tumor tissue relative to adjacent normal tissue. Furthermore, an increase in HURP expression was evident in all tumor samples relative to their respective normal tissue control with an average 3-fold increase ( p =0.001). Immunohistochemical analysis (IHC) of HURP revealed expression in all analyzed specimens. Although the stroma was weakly stained for HURP in some cases, all tumors were immunostained for this protein (average intensity score = ++, p p value = 0.387. Any conclusions about population-based distribution of poor prognosis with regards to ZEB1 positive CRC will require an adequately powered sample size. Overall, we have identified two stem cell-like molecules, HURP and ZEB1, with potential as markers of aggressiveness in CRC. Our findings also provide evidence of a possible biological basis for ethnicity-related differences with regards to stemness and regulatory factors of CRC aggressiveness. Citation Format: Logan Fair, Ingrid Espinoza, Xu Zhang, Abdelouahid Elkhattouti, Tangeng Ma, Joy King, Elizabeth Tarsi, Richard Whitlock, Vijay Kannuthurai, Ryan Jimenez, Tara Craft, Mary Graichen, Sharon Lobert, Roy Duhe, Charulochana Subramony, Christian R. Gomez, Christopher Lahr. HURP and ZEB1: Novel prognostic biomarkers in colorectal carcinomas. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B03.

Abstract 5143: MHC class I polypeptide-related sequence A (MICA) as a factor of aggressive prostate cancer

Prostate cancer (PCa) remains the second most common cancer in American men, with higher incidence and death rates in African American men (AAM) relative to Caucasian American men (CAM). MHC class I polypeptide-related sequence A (MICA) is a transmembrane protein responsible for activation of immunocytes, by binding the NKG2D receptor. The expression of MICA under normal conditions is low. However under stress conditions such as those found in neoplasms, MICA expression is increased. The soluble form of MICA (sMICA) is released by tumor cells and promotes immune evasion by binding and downregulating the NKG2D receptor in immune surveillance cells. Increased levels of sMICA have been found in patient sera of several types of cancer, including PCa. We hypothesized that low oxygen levels, a condition of the microenvironment present in aggressive tumors, would modify the levels of sMICA in PCa cell lines. Likewise we speculated that when compared to CAM, MICA would be differentially expressed in tumors from AAM, known to suffer a more aggressive disease. To address those questions, LNCaP cells were cultured under 20% O 2 (normoxia) and 2% O 2 (hypoxia) at various time points, supernatants were collected and the presence of sMICA in culture supernatant and patient sera was detected by ELISA. After 48 hours, the cells grown in hypoxia expressed about 5-fold more sMICA than cells grown in normoxia. When assessed in patient plasma, PCa patients had 2-fold more sMICA compared to healthy controls. In additional experiments cells growing under different oxygen levels were lysed and used in Western blot analysis to detect cellular MICA. Normal oxygen levels increased MICA protein by 3.5-fold. Hypoxia reduced MICA expression by 11%. A tissue microarray (TMA) was stained with MICA polyclonal antibody. The TMA was composed of 34 tumor samples and 30 controls. The samples came from 32 CAM and 32 AAM. The staining intensity was scored as 0, 1, 2 or 3 and scores 2 and 3 were defined as high intensity. Association of the staining result to presence of tumor was evaluated using the Fisher9s exact test. A higher proportion of high intensity in tumor site (50%) was found when compared to non-tumor site (20%, p = 0.019). Interestingly, stronger association was expressed in CAM (p = 0.036) than in AAM (p = 0.265). The results found in the cell line suggest that different oxygen levels affect the expression of sMICA. Higher expression of MICA in prostate tumor tissue and sMICA in patient sera are consistent with data previously reported in other cancers. Lower intensity in MICA staining in AAM patients may be related to better capacity of the tumor to evade the immune system. Overall our findings suggest the involvement of MICA on aggressive PCa. More studies are needed to decipher the biological mechanisms of MICA9s immune evasion in prostate tumors and to establish its contributing role to aggressive disease in minorities. Funding sources: PCRP W81XWH-14-1-0151, UMMC Office of Research Citation Format: Marcelo J. Sakiyama, Angel Garcia, Ingrid Espinoza, Jack Lewin, Xu Zhang, Elizabeth Tarsi, Tara Craft, Logan Fair, Jesus Monico, Lisa Sullivan, Charles R. Pound, Srinivasan Vijayakumar, Christian R. Gomez. MHC class I polypeptide-related sequence A (MICA) as a factor of aggressive 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 5143.

Abstract 1651: Overexpression of hepatoma upregulated protein (HURP) promotes radioresistance in prostate cancer cells

Despite progression in diagnosis and treatment, prostate cancer (PCa) still represents major cause of cancer-related mortality and morbidity in men. Although radiation therapy offers clinical benefit over other therapeutic modalities, the success of this therapeutic modality is commonly hampered by significant resistance in advanced tumor. So far, the mechanisms governing the acquired resistance to radiotherapy have not been discussed in detail. Hepatoma Up-Regulated Protein (HURP) is a cell cycle-regulated and microtubule-associated protein. It functions as a Ran GTPase effector and is involved in the stabilization of the mitotic kinetochore fibers. The expression of this protein is elevated in different tumor types and associated with tumor progression and aggressiveness. We recently reported on the value of HURP as an independent prognostic factor for high-risk PCa. The aim of this study was to address the role of HURP in the modulation of PCa resistance to γ- irradiation. Cell lines (LNCaP, PC3, and DU145) with regulated expression of HURP protein using a Lent viral Tet-On 3G Inducible Expression System were irradiated using a Cs-137 γ-source. Overexpression of HURP suppressed γ- irradiation- induced apoptosis. This suppression was associated with the expression of E2F1, p53, p21 together with the phosphorylation of apoptosis signal-regulating kinase1 (ASK1), c-jun-N-terminal kinase (JNK) and Ataxia-telangiectasia mutated (ATM) and histone family member X (H2AX). Also, inhibition of γ- irradiation- induced- cytochrome c release, cleavage of caspase-9, caspase-3, PARP, and reactive oxygen species (ROS) was noted. The observed resistance was consequence of HURP9s ability to trigger the ubiquitination of p53 and ATM that, in turn, resulted in the inhibition of downstream pathways, which are essential for the modulation of γ- irradiation-induced apoptosis. Our data provide evidence for the involvement of HURP in the modulation of PCa cell resistance to γ-irradiation via a mechanism mediated by ubiquitination of ATM and p53. Understanding of the biological mechanisms underlying HURP-mediated resistance of PCa to available therapies may help to improve the treatment strategy and emphasize HURP as therapeutic target for PCa treatment. Funding sources: Department of Defense Grant PC094680 and Prostate Cancer Foundation Creativity Award. Citation Format: Mohamed Hassan, Abdelouahid El Khattouti, Tangeng Ma, Ingrid Espinoza, W. Andrew Day, Srinivasan Vijayakumar, Christian R. Gomez. Overexpression of hepatoma upregulated protein (HURP) promotes radioresistance in prostate cancer cells. [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 1651.