Shu Wing Ng

SPARC (Secreted Protein Acidic and Rich in Cysteine) Induces Apoptosis in Ovarian Cancer Cells

Secreted protein acidic and rich in cysteine (SPARC) is an extracellular Ca(2+)-binding matricellular glycoprotein that associates with cell populations undergoing migration, morphogenesis, and differentiation. Studies on endothelial cells have established that its principal functions in vitro are counteradhesion and antiproliferation. The mechanism(s) underlying these antitumor effects is unknown. In this study, we showed that SPARC expression in ovarian cancer cells is inversely correlated with the degree of malignancy. The immunohistochemical data presented here confirmed the importance of diminished SPARC expression in ovarian cancer development. Treating human ovarian surface epithelial cells and ovarian cancer cells with SPARC revealed that as SPARC inhibits the proliferation of both normal and cancer cells, it induces apoptosis only in cancer cells. This observation indicates that down-regulation of SPARC is essential for ovarian carcinogenesis as cancer cells become sensitized to the apoptotic activity of SPARC during malignant transformation. We also showed here the first direct evidence that putative SPARC receptors are present on ovarian epithelial cells. Their levels are higher in human ovarian surface epithelial cells than cancer cells. Binding of SPARC to its receptor is likely to trigger tissue-specific signaling pathways that mediate its tumor suppressing functions. Decrease in ligand-receptor interaction by the down-regulation of SPARC and/or its receptor is essential for ovarian carcinogenesis.

DOC-2/hDab2, a candidate tumor suppressor gene involved in the development of gestational trophoblastic diseases

Gestational trophoblastic diseases comprise a spectrum of interrelated diseases including partial mole, complete mole and gestational choriocarcinoma. Using reverse transcriptase PCR (RT–PCR) analysis, we identified higher levels of DOC-2/hDab2 expression in the normal trophoblast cells in culture than in choriocarcinoma cell lines. Subsequent study using immunohistochemistry showed high levels of DOC-2/hDab2 protein expression in normal trophoblast tissues but significantly lower levels of expression in gestational trophoblastic disease tissues, particularly in complete mole and choriocarcinoma. When DOC-2/hDab2 was transfected into the choriocarcinoma cell lines, Jar, JEG and BeWo, the stable transfectants showed significantly reduced growth rate in culture. These data suggest that down regulation of DOC-2/hDab2 may play an important role in the development of gestational trophoblastic diseases.

Selenium binding protein 1 in ovarian cancer

Selenium binding protein 1 (SELENBP1) was identified to be the most significantly down‐regulated protein in ovarian cancer cells by a membrane proteome profiling analysis. SELENBP1 expression levels in 4 normal ovaries, 8 benign ovarian tumors, 12 borderline ovarian tumors and 141 invasive ovarian cancers were analyzed with immunohistochemical assay. SELENBP1 expression was reduced in 87% cases of invasive ovarian cancer (122/141) and was significantly reduced in borderline tumors and invasive cancers (p < 0.001). Cox multivariate analysis within the 141 invasive cancer tissues showed that SELENBP1 expression score was a potential prognostic indicator for unfavorable prognosis of ovarian cancer (hazard ratio [HR], 2.18; 95% CI = 1.22–3.90; p = 0.009). Selenium can disrupt the androgen pathway, which has been implicated in modulating SELENBP1 expression. We investigated the effects of selenium and androgen on normal human ovarian surface epithelial (HOSE) cells and cancer cells. Interestingly, SELENBP1 mRNA and protein levels were reduced by androgen and elevated by selenium treatment in the normal HOSE cells, whereas reversed responses were observed in the ovarian cancer cell lines. These results suggest that changes of SELENBP1 expression in malignant ovarian cancer are an indicator of aberration of selenium/androgen pathways and may reveal prognostic information of ovarian cancer.

Analysis of p73 in human borderline and invasive ovarian tumor.

p73 is a novel gene that has high sequence homology and similar gene structure to the tumor suppressor gene p53. We analysed p73 in seven ovarian carcinoma cell lines and a total of 63 human borderline and invasive ovarian tumor samples. Loss of heterozygosity at this locus was observed in 50% of invasive tumors but in none of the borderline tumors. Biallelic expression of the gene was observed in the heterozygous tumor tissues. Direct sequencing and single-strand conformation polymorphism analyses of the p73 cDNA sequence homologous to the highly mutatable region of p53 did not reveal any mutations. When compared to the primary cultures of normal human ovarian surface epithelial cells and immortalized cell lines, four of the seven ovarian carcinoma cell lines, 71% of the invasive tumors, and 92% of the borderline tumor tissues express elevated levels of p73 transcript. Except for the OVCA3 cell line, Western blot analysis of the nuclear extracts prepared from the cell lines showed concordant levels of p73 protein. Our analysis also demonstrated the expression of a spliced variant of p73 transcript with the omission of exon 2 solely in the cancer cell lines and invasive tumor tissues. This exon 2-spliced transcript would give rise to a truncated p73 protein without the N-terminal transactivation domain. In reminiscence of the dominant negative phenotype of the N-terminal truncated variants of another p53-related gene, p63, the expression of the truncated p73 variant form in ovarian tumors may play an important role in the pathogenesis of ovarian cancer.

Matrix metalloproteinases and their inhibitors and inducer in gestational trophoblastic diseases and normal placenta

OBJECTIVES This study aimed to investigate the expression of recently identified matrix metalloproteinases (MMPs), their inhibitors (TIMPs), and inducer (CD147) in a wide range of gestational trophoblastic diseases (GTD) thereby expanding our understanding of the potential role of MMPs in GTD. METHODS Paraffin sections of 10 normal first-trimester placentas (NP), 10 partial moles (PM), 10 complete moles (CM), 5 choriocarcinomas (CCA) and 5 placental site trophoblastic tumors (PSTT) were studied immunohistochemically for expression of MMP-7, MMP-14, MMP-21, MMP-28, TIMP-3, TIMP-4 and CD147. Immunolocalization of MMP-1, MMP-2, MMP-3, MMP-9, MMP-13 and TIMP-1 was performed on 5 CCA and 5 PSTTs. RESULTS CCA showed stronger intensity for MMP-14 and MMP-28 than PSTT (p<0.05, p<0.05). CCA and PSTT had stronger expression of MMP-21 than NP, PM and CM (p<0.05, p<0.05, p<0.01). PSTT (p<0.05, p<0.05), NP (p<0.01, p<0.01) and CM (p<0.01, p<0.05) showed stronger staining for TIMP-3 and TIMP-4 than CCA. CONCLUSION Choriocarcinomas high expression of MMPs and low expression of MMP inhibitors may contribute to its invasiveness and metastatic potential. Similarly, PSTTs lower expression of MMPs and high expression of MMP inhibitors may partly explain its lower invasiveness. Agents that inhibit MMP may prove useful in treating GTD.

Differential expression of NF1 type I and type II isoforms in sporadic borderline and invasive epithelial ovarian tumors

The NF1 gene, a putative tumor suppressor gene, contains a GAP related domain (GRD) which accelerates hydrolysis of ras-bound GTP to GDP, thereby converting the ras oncogene from its active to inactive form. Two forms of the NF1 GRD transcript (Type I and Type II) are differentially expressed in neuroectodermal tumor tissue relative to differentiated neural cells, and in gastric cancer cell lines relative to normal stomach mucosa. We measured relative expression of NF1 Type II and Type I isoforms in cultured normal and malignant human ovarian surface epithelial cells(HOSE) and in invasive and borderline ovarian tumor tissue. We demonstrated an 11-fold increase in Type II: Type I ratio in 7 HOSE cultures relative to eight ovarian cancer cell lines. Our findings indicate a significant decrease in Type II isoform expression and increase in Type I expression in ovarian cancer cells and tumor tissue relative to HOSE cells. We also demonstrate an increase in Type II: Type I ratio, and a decrease in cell proliferation rate in three ovarian cancer cell lines on treatment with retinoic acid. We propose that differential expression of the NF1 Type I and Type II isoforms is related to cellular differentiation in ovarian epithelial cancer and strategies based on alteration in NF1 isoform expression may have thera-peutic potential in ovarian malignancies.

Protein Kinases and Associated Pathways in Pluripotent State and Lineage Differentiation

Protein kinases (PKs) mediate the reversible conversion of substrate proteins to phosphorylated forms, a key process in controlling intracellular signaling transduction cascades. Pluripotency is, among others, characterized by specifically expressed PKs forming a highly interconnected regulatory network that culminates in a finely-balanced molecular switch. Current high-throughput phosphoproteomic approaches have shed light on the specific regulatory PKs and their function in controlling pluripotent states. Pluripotent cell-derived endothelial and hematopoietic developments represent an example of the importance of pluripotency in cancer therapeutics and organ regeneration. This review attempts to provide the hitherto known kinome profile and the individual characterization of PK-related pathways that regulate pluripotency. Elucidating the underlying intrinsic and extrinsic signals may improve our understanding of the different pluripotent states, the maintenance or induction of pluripotency, and the ability to tailor lineage differentiation, with a particular focus on endothelial cell differentiation for anti-cancer treatment, cell-based tissue engineering, and regenerative medicine strategies.

Abstract MIP-052: THE ROLES OF CD24 IN OVARIAN CANCER DEVELOPMENT

OBJECTIVES: CD24 is recently reported as ovarian cancer stem cell markers, and the expression of CD24 is increased from borderline tumors to invasive ovarian carcinomas. However, the underlying pathways controlled by CD24 in ovarian pathogenesis were poorly understood. We investigated the potential roles of CD24 in normal fallopian tube epithelial (FTE) cells and ovarian cancer development. METHODS: Lentiviral particles harboring CD24 shRNAs targeting CD24 expression were used to establish FTE cell lines and ovarian cancer cell lines with CD24 knockdown. The resulting cell lines were compared with the correspondent cells with non target shRNA control in colony formation assay, BrdU incorporation, cell growth and drug resistance. Western blot was used for analyzing downstream pathways affected by the knockdown of CD24. Expression of CD24 and its downstream effectors identified from western blot, including Pax 2 and Gli1 were studied in normal FTE, non–serous and serous tumor tissues by immunohistochemistry (IHC) to confirm whether the in vitro study is clinically relevant. RESULTS: Western blot analysis showed that CD24 expression was absent in normal ovarian surface epithelial (OSE) cells but was relatively high in normal FTE cells. In addition, serous cancer cell lines have a lower CD24 expression when compared to non–serous cancer cell lines. Knockdown of CD24 in FTE cells caused slower growth, lower ability in colony formation, reduced percentage of cells in S phase and decrease in expression of p–stat3 in JAK pathway and the JAK downstream targets Nanog and OCT3/4; and Pax2, which is normally expressed in FTE but lost in FTE secretory cell outgrowth (SCOUT). The loss of Pax2 in SCOUT is associated with serous carcinoma. IHC showed cytoplasmic co–localized expression of CD24 and Pax2 in the normal FTE tissue, suggesting the possibility of interaction between these two proteins in vivo. In contrast, knockdown of CD24 in ovarian cancer cells caused faster growth, higher ability in colony formation, increased percentage of cells in S phase, more resistant to carboplatin and higher expression of Shh and Gli1 in Sonic hedgehog (SHH) pathway. The above effects were more obvious in the non–serous cancer cell line. Activation of SHH pathway requires translocation of Gli1 into the nucleus. Concurrent with the in vitro results, IHC showed that the expression of Gli1 was found inside the nucleus in the non–serous tumor tissues but in the cytoplasm in serous–tissue, suggesting that the effect brought by SHH pathway is more prominent in the non–serous cancer cells. CONCLUSION: Our study is the first study revealing the linkage between CD24 and PAX2, and suggesting normal FTE cells depends on CD24/JAK pathway for growth and the loss of CD24 expression and hence, the loss of PAX2 may be associated with serous type tumor development, basing on other studies that SCOUTs were significantly associated with serous carcinoma. Moreover, the study also suggests CD24 pathway affects Type I and Type II (HGSOC) tumors differently. The CD24/SHH pathways may be responsible in controlling cell growth and drug resistance in ovarian cancer cells, hence, warrant further studies. Citation Format: Pui–Wah Choi, Brooke E. Howitt, Christopher P. Crum, Ross S. Berkowitz, and Shu–Wing Ng. THE ROLES OF CD24 IN OVARIAN CANCER DEVELOPMENT [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-052.

Abstract A23: The dual roles of microRNA-200: From inclusion cyst formation to cell migration.

Background: Historically, metaplasia of ovarian surface epithelial (OSE) cells in the cortical inclusion cysts is associated with ovarian cancer development. Recent studies have also suggested fallopian tube epithelium as another origin of ovarian cancer cells. MicroRNA-200 (miR200) family is related to epithelial-mesenchymal transition (EMT) and overexpressed in ovarian cancer. However, the potential involvement of miR200 in ovarian pathogenesis is largely unknown. Objective: To study any morphological and signaling differences between immortalized normal human ovarian surface epithelial (OSE) cells and ovarian cancer cells with changes in miR200 expression and control cells growing in three-dimensional (3D) cultures. Methods: OSE cells with stable ectopic expression of miR200 and ovarian cancer cells with stable miR200 knockdown were established using lentiviral agents. The resulting cells and control cells were grown as 3D cultures first in Matrigel to form the cyst-like spheroids and then transferred to Collagen I matrix for cell migration study. Immunofluorescence and confocal microscopy was applied to study the morphologies of the 3D structures, and in conjunction with Western blot analysis, to study changes in signaling pathways in response to changes of miR200 expression and in the presence of a variety of pharmacologic inhibitors. Clinical samples were examined for miR200 expression to evaluate the results from 3D in vitro cultures. Results: In comparison with the wild-type control counterparts, OSE cells with ectopic expression of miR200 had elevated E-cadherin expression, showed more compact and prolonged cyst stabilization in both Matrigel and Collagen I 3D cultures; whereas the ovarian cancer cells with miR200 knockdown showed increased TGFβ expression and multiple lumens with enhanced cell death in Matrigel culture, and change from collective migration mode to single cells in Collagen I culture. Both acetylated tubulin and Histone H3 staining of mitotic cells in Matrigel culture demonstrated ovarian cancer cells with miR200 knockdown had abnormal spindle orientation and defective cell cycle division. Application of TGFβ inhibitors reversed the single cell migration phenotype of miR200 knockdown cancer cells to collective movement. However, the downstream pathways responsible for the switching of migration phenotype were not the same between the serous and mucinous cell lines. For the mucinous cell line, miR200 controlled the switching through TGFβ-ROCK pathway, whereas the downstream pathway for the switching in the serous cell line remained unknown. qRT-PCR analysis of micro-dissected cells from clinical samples verified the elevated expression of miR200 in endosalpingiosis cysts and tumor tissues when compared with normal ovarian surface epithelia and fallopian tube epithelia. Conclusion: The qRT-PCR validation study verifies the findings of our 3D in vitro culture study and demonstrates that miR200 is important for inclusion cyst formation and collective movement of ovarian cancer cells during tumor invasion. Perturbation of this pathway in cancer cells will accelerate cell death through abnormal cell division. Hence, miR200 may be an interesting target in ovarian cancer therapy. Citation Format: Pui Wah Choi, Junzheng Yang, Wing Ping Fong, Ross S. Berkowitz, William R. Welch, Gregory J. Goodall, Shu Wing Ng. The dual roles of microRNA-200: From inclusion cyst formation to cell migration. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A23.