Knockdown of stem cell regulator Oct4A in ovarian cancer reveals cellular reprogramming associated with key regulators of cytoskeleton-extracellular matrix remodelling
- Samardzija, Chantel, Greening, David, Escalona, Ruth, Chen, Maoshan, Bilandzic, Maree, Luwor, Rodney, Kannourakis, George, Findlay, Jock, Ahmed, Nuzhat
- Authors: Samardzija, Chantel , Greening, David , Escalona, Ruth , Chen, Maoshan , Bilandzic, Maree , Luwor, Rodney , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2017
- Type: Text , Journal article
- Relation: Scientific Reports Vol. 7, no. (2017), p. 1-18
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- Description: Oct4A is a master regulator of self-renewal and pluripotency in embryonic stem cells. It is a well-established marker for cancer stem cell (CSC) in malignancies. Recently, using a loss of function studies, we have demonstrated key roles for Oct4A in tumor cell survival, metastasis and chemoresistance in in vitro and in vivo models of ovarian cancer. In an effort to understand the regulatory role of Oct4A in tumor biology, we employed the use of an ovarian cancer shRNA Oct4A knockdown cell line (HEY Oct4A KD) and a global mass spectrometry (MS)-based proteomic analysis to investigate novel biological targets of Oct4A in HEY samples (cell lysates, secretomes and mouse tumor xenografts). Based on significant differential expression, pathway and protein network analyses, and comprehensive literature search we identified key proteins involved with biologically relevant functions of Oct4A in tumor biology. Across all preparations of HEY Oct4A KD samples significant alterations in protein networks associated with cytoskeleton, extracellular matrix (ECM), proliferation, adhesion, metabolism, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and drug resistance was observed. This comprehensive proteomics study for the first time presents the Oct4A associated proteome and expands our understanding on the biological role of this stem cell regulator in carcinomas. © 2017 The Author(s).
- Authors: Samardzija, Chantel , Greening, David , Escalona, Ruth , Chen, Maoshan , Bilandzic, Maree , Luwor, Rodney , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2017
- Type: Text , Journal article
- Relation: Scientific Reports Vol. 7, no. (2017), p. 1-18
- Full Text:
- Reviewed:
- Description: Oct4A is a master regulator of self-renewal and pluripotency in embryonic stem cells. It is a well-established marker for cancer stem cell (CSC) in malignancies. Recently, using a loss of function studies, we have demonstrated key roles for Oct4A in tumor cell survival, metastasis and chemoresistance in in vitro and in vivo models of ovarian cancer. In an effort to understand the regulatory role of Oct4A in tumor biology, we employed the use of an ovarian cancer shRNA Oct4A knockdown cell line (HEY Oct4A KD) and a global mass spectrometry (MS)-based proteomic analysis to investigate novel biological targets of Oct4A in HEY samples (cell lysates, secretomes and mouse tumor xenografts). Based on significant differential expression, pathway and protein network analyses, and comprehensive literature search we identified key proteins involved with biologically relevant functions of Oct4A in tumor biology. Across all preparations of HEY Oct4A KD samples significant alterations in protein networks associated with cytoskeleton, extracellular matrix (ECM), proliferation, adhesion, metabolism, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and drug resistance was observed. This comprehensive proteomics study for the first time presents the Oct4A associated proteome and expands our understanding on the biological role of this stem cell regulator in carcinomas. © 2017 The Author(s).
TIMP-2 regulates proliferation, invasion and STAT3-mediated cancer stem cell-dependent chemoresistance in ovarian cancer cells
- Escalona, Ruth, Bilandzic, Maree, Western, Patrick, Kadife, Elif, Kannourakis, George, Findlay, Jock, Ahmed, Nuzhat
- Authors: Escalona, Ruth , Bilandzic, Maree , Western, Patrick , Kadife, Elif , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2020
- Type: Text , Journal article
- Relation: BMC Cancer Vol. 20, no. 1 (2020), p.
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- Description: Background: The metzincin family of metalloproteinases and the tissue inhibitors of metalloproteinases (TIMPs) are essential proteins required for biological processes during cancer progression. This study aimed to determine the role of TIMP-2 in ovarian cancer progression and chemoresistance by reducing TIMP-2 expression in vitro in Fallopian tube secretory epithelial (FT282) and ovarian cancer (JHOS2 and OVCAR4) cell lines. Methods: FT282, JHOS2 and OVCAR4 cells were transiently transfected with either single or pooled TIMP-2 siRNAs. The expression of different genes after TIMP-2 knock down (T2-KD) or in response to chemotherapy was determined at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence. Sensitivity of the cell lines in response to chemotherapy after TIMP-2 knock down was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Ethynyl-2′-deoxyuridine (EdU) assays. Cell invasion in response to TIMP-2 knockdown was determined by xCELLigence. Results: Sixty to 90 % knock down of TIMP-2 expression was confirmed in FT282, OVCAR4 and JHOS2 cell lines at the mRNA and protein levels. TIMP-2 knock down did not change the mRNA expression of TIMP-1 or TIMP-3. However, a significant downregulation of MMP-2 in T2-KD cells occurred at both the protein and activation levels, compared to Control (Cont; scrambled siRNA) and Parental cells (P, transfection reagent only). In contrast, membrane bound MT1-MMP protein levels were significantly upregulated in T2-KD compared to Cont and P cells. T2-KD cells exhibited enhanced proliferation and increased sensitivity to cisplatin and paclitaxel treatments. Enhanced invasion was observed in the T2-KD-JOSH2 and OVCAR4 cells but not in T2-KD-FT282 cells. Treatment with cisplatin or paclitaxel significantly elevated the expression of TIMP-2 in Cont cells but not in T2-KD cells, consistent with significantly elevated expression of chemoresistance and CSC markers and activation of STAT3. Furthermore, a potent inhibitor of STAT3 activation, Momelotinib, suppressed chemotherapy-induced activation of P-STAT3 in OVCAR4 cells with concomitant reductions in the expression of chemoresistance genes and CSC markers. Conclusions: The above results suggest that TIMP-2 may have a novel role in ovarian cancer proliferation, invasion and chemoresistance. © 2020 The Author(s).
- Authors: Escalona, Ruth , Bilandzic, Maree , Western, Patrick , Kadife, Elif , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2020
- Type: Text , Journal article
- Relation: BMC Cancer Vol. 20, no. 1 (2020), p.
- Full Text:
- Reviewed:
- Description: Background: The metzincin family of metalloproteinases and the tissue inhibitors of metalloproteinases (TIMPs) are essential proteins required for biological processes during cancer progression. This study aimed to determine the role of TIMP-2 in ovarian cancer progression and chemoresistance by reducing TIMP-2 expression in vitro in Fallopian tube secretory epithelial (FT282) and ovarian cancer (JHOS2 and OVCAR4) cell lines. Methods: FT282, JHOS2 and OVCAR4 cells were transiently transfected with either single or pooled TIMP-2 siRNAs. The expression of different genes after TIMP-2 knock down (T2-KD) or in response to chemotherapy was determined at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence. Sensitivity of the cell lines in response to chemotherapy after TIMP-2 knock down was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Ethynyl-2′-deoxyuridine (EdU) assays. Cell invasion in response to TIMP-2 knockdown was determined by xCELLigence. Results: Sixty to 90 % knock down of TIMP-2 expression was confirmed in FT282, OVCAR4 and JHOS2 cell lines at the mRNA and protein levels. TIMP-2 knock down did not change the mRNA expression of TIMP-1 or TIMP-3. However, a significant downregulation of MMP-2 in T2-KD cells occurred at both the protein and activation levels, compared to Control (Cont; scrambled siRNA) and Parental cells (P, transfection reagent only). In contrast, membrane bound MT1-MMP protein levels were significantly upregulated in T2-KD compared to Cont and P cells. T2-KD cells exhibited enhanced proliferation and increased sensitivity to cisplatin and paclitaxel treatments. Enhanced invasion was observed in the T2-KD-JOSH2 and OVCAR4 cells but not in T2-KD-FT282 cells. Treatment with cisplatin or paclitaxel significantly elevated the expression of TIMP-2 in Cont cells but not in T2-KD cells, consistent with significantly elevated expression of chemoresistance and CSC markers and activation of STAT3. Furthermore, a potent inhibitor of STAT3 activation, Momelotinib, suppressed chemotherapy-induced activation of P-STAT3 in OVCAR4 cells with concomitant reductions in the expression of chemoresistance genes and CSC markers. Conclusions: The above results suggest that TIMP-2 may have a novel role in ovarian cancer proliferation, invasion and chemoresistance. © 2020 The Author(s).
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