Imelda Mercado-Uribe

The chemokine growth-regulated oncogene 1 (Gro-1) links RAS signaling to the senescence of stromal fibroblasts and ovarian tumorigenesis

Epithelial–stromal interactions play a critical role in tumor initiation and progression; cancer-associated stroma, but not normal stroma, is known to be tumor-promoting. However, the molecular signal used by epithelial cancer cells to reprogram normal stroma to a tumorigenic stroma is not known. Here, we present evidence to suggest that the chemokine growth-regulated oncogene 1 (Gro-1) may be one such signaling molecule. We showed that the expression of Gro-1 is activated by RAS and is vital for cell survival and the malignant transformation of ovarian epithelial cells. Surprisingly, we found that Gro-1 is a potent inducer of senescence in stromal fibroblasts and that this effect depends on functional p53. Senescent fibroblasts induced by Gro-1 can promote tumor growth whereas abrogation of senescence through immortalization results in loss of such tumor promoting activity. We also demonstrated that stromal fibroblasts adjacent to epithelial cancer cells are senescent in human ovarian cancer specimens and in heterografts from RAS-transformed human ovarian epithelial cells and ovarian cancer cells. Moreover, Gro-1 was expressed at significantly higher amounts in ovarian cancer than in normal tissues and was higher in serum samples from women with ovarian cancer than in serum from women without ovarian cancer. These findings provide strong evidence that RAS-induced Gro-1 can reprogram the stromal microenvironment through the induction of senescence of fibroblasts and thus can promote tumorigenesis. Therefore, Gro-1 may be a therapeutic target as well as a diagnostic marker in ovarian cancer.

Generation of cancer stem-like cells through the formation of polyploid giant cancer cells

Polyploid giant cancer cells (PGCCs) have been observed by pathologists for over a century. PGCCs contribute to solid tumor heterogeneity, but their functions are largely undefined. Little attention has been given to these cells, largely because PGCCs have been generally thought to originate from repeated failure of mitosis/cytokinesis and have no capacity for long-term survival or proliferation. Here we report our successful purification and culture of PGCCs from human ovarian cancer cell lines and primary ovarian cancer. These cells are highly resistant to oxygen deprivation and could form through endoreduplication or cell fusion, generating regular-sized cancer cells quickly through budding or bursting similar to simple organisms like fungi. They express normal and cancer stem cell markers, they divide asymmetrically and they cycle slowly. They can differentiate into adipose, cartilage and bone. A single PGCC formed cancer spheroids in vitro and generated tumors in immunodeficient mice. These PGCC-derived tumors gained a mesenchymal phenotype with increased expression of cancer stem cell markers CD44 and CD133 and become more resistant to treatment with cisplatin. Taken together, our results reveal that PGCCs represent a resistant form of human cancer using an ancient, evolutionarily conserved mechanism in response to hypoxia stress; they can contribute to the generation of cancer stem-like cells, and also play a fundamental role in regulating tumor heterogeneity, tumor growth and chemoresistance in human cancer.

The role of constitutively active signal transducer and activator of transcription 3 in ovarian tumorigenesis and prognosis

Signal transducer and activator of transcription 3 (Stat3), which is a latent transcription factor that participates in the transcriptional activation of apoptosis and cell cycle progression, has been implicated as an oncogene in several neoplastic diseases. However, the specific role of Stat3 in ovarian carcinogenesis remains poorly understood. The objectives of the current study were to examine the effect of Stat3 activation on the phenotypic transformation of an immortalized, nontumorigenic ovarian epithelial cell line and to evaluate the expression of tyrosine‐activated Stat3 (pStat3) in tissue microarrays from 303 ovarian carcinomas to determine its prognostic relevance and to correlate its expression with several upstream oncogenes of Stat3 and with the oncogenes involved in apoptosis and proliferation.

CXCR2 Promotes Ovarian Cancer Growth through Dysregulated Cell Cycle, Diminished Apoptosis, and Enhanced Angiogenesis

Purpose: Chemokine receptor CXCR2 is associated with malignancy in several cancer models; however, the mechanisms involved in CXCR2-mediated tumor growth remain elusive. Here, we investigated the role of CXCR2 in human ovarian cancer. Experimental Design: CXCR2 expression was silenced by stable small hairpin RNA in ovarian cancer cell lines T29Gro-1, T29H, and SKOV3. Western blotting, immunofluorescence, enzyme-linked immunosorbent assay, flow cytometry, electrophoretic mobility shift assay, and mouse assay were used to detect CXCR2, interleukin-8, Gro-1, cell cycle, apoptosis, DNA binding of NF-κB, and tumor growth. Immunohistochemical staining of CXCR2 was done in 240 high-grade serous ovarian carcinoma samples. Results: Knockdown of CXCR2 expression by small hairpin RNA reduced tumorigenesis of ovarian cancer cells in nude mice. CXCR2 promoted cell cycle progression by modulating cell cycle regulatory proteins, including p21 (waf1/cip1), cyclin D1, CDK6, CDK4, cyclin A, and cyclin B1. CXCR2 inhibited cellular apoptosis by suppressing phosphorylated p53, Puma, and Bcl-xS; suppressing poly(ADP-ribose) polymerase cleavage; and activating Bcl-xL and Bcl-2. CXCR2 stimulated angiogenesis by increasing levels of vascular endothelial growth factor and decreasing levels of thrombospondin-1, a process likely involving mitogen-activated protein kinase, and NF-κB. Overexpression of CXCR2 in high-grade serous ovarian carcinomas was an independent prognostic factor of poor overall survival (P < 0.001) and of early relapse (P = 0.003) in the univariate analysis. Conclusions: Our data provide strong evidence that CXCR2 regulates the cell cycle, apoptosis, and angiogenesis through multiple signaling pathways, including mitogen-activated protein kinase and NF-κB, in ovarian cancer. CXCR2 thus has potential as a therapeutic target and for use in ovarian cancer diagnosis and prognosis. Clin Cancer Res; 16(15); 3875–86. ©2010 AACR.

Metabolic shifts toward glutamine regulate tumor growth, invasion and bioenergetics in ovarian cancer

Glutamine can play a critical role in cellular growth in multiple cancers. Glutamine‐addicted cancer cells are dependent on glutamine for viability, and their metabolism is reprogrammed for glutamine utilization through the tricarboxylic acid (TCA) cycle. Here, we have uncovered a missing link between cancer invasiveness and glutamine dependence. Using isotope tracer and bioenergetic analysis, we found that low‐invasive ovarian cancer (OVCA) cells are glutamine independent, whereas high‐invasive OVCA cells are markedly glutamine dependent. Consistent with our findings, OVCA patients’ microarray data suggest that glutaminolysis correlates with poor survival. Notably, the ratio of gene expression associated with glutamine anabolism versus catabolism has emerged as a novel biomarker for patient prognosis. Significantly, we found that glutamine regulates the activation of STAT3, a mediator of signaling pathways which regulates cancer hallmarks in invasive OVCA cells. Our findings suggest that a combined approach of targeting high‐invasive OVCA cells by blocking glutamines entry into the TCA cycle, along with targeting low‐invasive OVCA cells by inhibiting glutamine synthesis and STAT3 may lead to potential therapeutic approaches for treating OVCAs.

CD133 expression associated with poor prognosis in ovarian cancer

As a putative marker for cancer stem cells in human malignant tumors, including ovarian cancer, CD133 expression may define a tumor-initiating subpopulation of cells and is associated with the clinical outcome of patients. However, at this time its clinical significance in ovarian cancer remains uncertain. The aim of this study was to clarify the clinical role of CD133 expression in human ovarian cancer. Immunohistochemical staining of CD133 expression was performed in 400 ovarian carcinoma samples using tissue microarray. The associations among CD133 expression and clinical factors (diagnosis, tumor grade, cancer stage, and clinical response to chemotherapy), overall survival and disease-free survival time were analyzed. CD133 expression was found in 31% of ovarian carcinoma samples. Fishers exact test and one-way analysis of variance suggested that CD133 expression was associated with high-grade serous carcinoma (P=0.035), late-stage disease (P<0.001), ascites level (P=0.010), and non-response to chemotherapy (P=0.023). CD133 expression was also associated with shorter overall survival time (P=0.007) and shorter disease-free survival time (P<0.001) by log-rank test. Moreover, CD133 expression was an independent predictor of shorter disease-free survival time in an unconditional logistic regression analysis with multiple covariates (P=0.024). Our results thus show that CD133 expression is a predictor of poor clinical outcome for patients with ovarian cancer, supporting the proposed link between CD133 and cancer stem cells.

Stanniocalcin 1 and Ovarian Tumorigenesis

BACKGROUND Stanniocalcin 1 (STC1) is a secreted glycoprotein hormone. High expression of STC1 has been associated with several cancers including ovarian cancer, but its role in the development of ovarian cancer is not clear. METHODS We used five human ovarian epithelial cancer cell lines (OVCA420, OVCA432, OVCA433, SKOV3, and HEY), immortalized human ovarian surface epithelial cells (T29 and T80), ovarian cancer tissues from 342 patients, serum from 73 ovarian cancer patients and from58 control subjects, and 116 mice, with six or eight per group. Protein expression was assessed. Cells overexpressing STC1 protein were generated by ectopic expression of human STC1 cDNA. STC1 expression was silenced by using small interfering RNA against STC1. Cell proliferation, migration, colony formation, and apoptosis were assessed. Xenograft tumor growth in mice was studied. Neutralizing anti-STC1 antibody was used to inhibit STC1 function. All statistical tests were two-sided. RESULTS STC1 protein expression was higher in all human ovarian cancer cell lines examined than in immortalized human ovarian epithelial cell lines, higher in ovarian cancer tissue than in normal ovarian tissue (P < .001), and higher in serum from ovarian cancer patients than from control subjects (P = .021). Ovarian cancer cells with STC1 overexpression, compared with corresponding control cells, had increased cell proliferation, migration, and colony formation in cell culture and increased growth of xenograft tumors in mice. These activities in normal or malignant ovarian cells with STC1 overexpression, compared with control cells, were also accompanied by increased expression of cell cycle regulatory proteins and antiapoptotic proteins but decreased cleavage of several caspases. Within 24 hours of treatment, apoptosis in cultures of HEY ovarian cancer cells treated with neutralizing anti-STC1 monoclonal antibody was higher (17.3% apoptotic cells) than that in cultures treated with mouse IgG control cells (4.4%) (12.9% difference, 95% confidence interval = 11.6% to 14.2%). CONCLUSIONS STC1 protein may be involved in ovarian tumorigenesis.

The Biphasic Role of NF-κB in Progression and Chemoresistance of Ovarian Cancer

Purpose: NF-κB is a transcription factor known to promote tumorigenesis. However, NF-κB is also known to be proapoptotic and may potentially function as a tumor suppressor, although such a functional role has not been extensively investigated in human cancer. Experimental Design: A dominant-negative mutant of IκBα with mutations at S32A and S36A was used to inhibit the function of NF-κB in ovarian cancer cell lines. The transcription ability, tumorigenesis, apoptosis, and drug sensitivity were examined in derivative cell lines in comparison with parental cells. We also analyzed the association of nuclear expression of NF-κB p65 with patient survival in an ovarian cancer tissue array. Results: We show that NF-κB functions as a tumor suppressor in four ovarian cancer cell lines, but it functions as an oncogene in their aggressive chemoresistant isogenic variants. NF-κB can exert its proapoptotic or antiapoptotic effect by activating or repressing mitogen-activated protein kinase (MAPK) phosphorylation in parental or aggressive chemoresistant variant cell lines. We also show that the nuclear accumulation of p65 in epithelial cancer tissue is associated with a good response to chemotherapy and can predict longer overall survival for patients with ovarian cancer. Conclusions: Our data provide strong evidence that NF-κB can function as a biphasic regulator, either suppressing or enhancing ovarian cancer growth through the regulation of MAPK and cellular apoptosis. Clin Cancer Res; 17(8); 2181–94. ©2011 AACR.

Aurora Kinase A Promotes Ovarian Tumorigenesis through Dysregulation of the Cell Cycle and Suppression of BRCA2

Purpose: Aurora kinase A (Aurora-A) is known to regulate genomic instability and tumorigenesis in multiple human cancers. The underlying mechanism, however, is not fully understood. We examined the molecular mechanism of Aurora-A regulation in human ovarian cancer. Experimental Design: Retrovirus-mediated small hairpin RNA (shRNA) was used to silence the expression of Aurora-A in the ovarian cancer cell lines SKOV3, OVCA432, and OVCA433. Immunofluorescence, Western blotting, flow cytometry, cytogenetic analysis, and animal assay were used to test centrosome amplification, cell cycle alteration, apoptosis, DNA damage response, tumor growth, and genomic instability. Immunostaining of BRCA2 and Aurora-A was done in ovarian, pancreatic, breast, and colon cancer samples. Results: Knockdown of Aurora-A reduced centrosome amplification, malformation of mitotic spindles, and chromosome aberration, leading to decreased tumor growth. Silencing Aurora-A attenuated cell cycle progression and enhanced apoptosis and DNA damage response by restoring p21, pRb, and BRCA2 expression. Aurora-A was inversely correlated with BRCA2 in high-grade ovarian serous carcinoma, breast cancer, and pancreatic cancer. In high-grade ovarian serous carcinoma, positive expression of BRCA2 predicted increased overall and disease-free survival, whereas positive expression of Aurora-A predicted poor overall and disease-free survival (P < 0.05). Moreover, an increased Aurora-A to BRCA2 expression ratio predicted poor overall survival (P = 0.047) compared with a decreased Aurora-A to BRCA2 expression ratio. Conclusion: Aurora-A regulates genomic instability and tumorigenesis through cell cycle dysregulation and BRCA2 suppression. The negative correlation between Aurora-A and BRCA2 exists in multiple cancers, whereas the expression ratio of Aurora-A to BRCA2 predicts ovarian cancer patient outcome. Clin Cancer Res; 16(12); 3171–81. ©2010 AACR.

Ovarian cancer: pathology, biology, and disease models.

Epithelial ovarian cancer, which comprises several histologic types and grades, is the most lethal cancer among women in the United States. In this review, we summarize recent progress in understanding the pathology and biology of this disease and in development of models for preclinical research. Our new understanding of this disease suggests new targets for therapeutic intervention and novel markers for early detection of disease.