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).
The many facets of metzincins and their endogenous inhibitors : Perspectives on ovarian cancer progression
- Escalona, Ruth, Chan, Emily, Kannourakis, George, Findlay, Jock, Ahmed, Nuzhat
- Authors: Escalona, Ruth , Chan, Emily , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2018
- Type: Text , Journal article , Review
- Relation: International Journal of Molecular Sciences Vol. 19, no. 2 (2018), p. 1-33
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- Description: Approximately sixty per cent of ovarian cancer patients die within the first five years of diagnosis due to recurrence associated with chemoresistance. The metzincin family of metalloproteinases is enzymes involved in matrix remodeling in response to normal physiological changes and diseased states. Recently, there has been a mounting awareness of these proteinases and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), as superb modulators of cellular communication and signaling regulating key biological processes in cancer progression. This review investigates the role of metzincins and their inhibitors in ovarian cancer. We propose that understanding the metzincins and TIMP biology in ovarian cancer may provide valuable insights in combating ovarian cancer progression and chemoresistance-mediated recurrence in patients. © 2018 by the authors. Licensee MDPI, Basel, Switzerland.
- Authors: Escalona, Ruth , Chan, Emily , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2018
- Type: Text , Journal article , Review
- Relation: International Journal of Molecular Sciences Vol. 19, no. 2 (2018), p. 1-33
- Full Text:
- Reviewed:
- Description: Approximately sixty per cent of ovarian cancer patients die within the first five years of diagnosis due to recurrence associated with chemoresistance. The metzincin family of metalloproteinases is enzymes involved in matrix remodeling in response to normal physiological changes and diseased states. Recently, there has been a mounting awareness of these proteinases and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), as superb modulators of cellular communication and signaling regulating key biological processes in cancer progression. This review investigates the role of metzincins and their inhibitors in ovarian cancer. We propose that understanding the metzincins and TIMP biology in ovarian cancer may provide valuable insights in combating ovarian cancer progression and chemoresistance-mediated recurrence in patients. © 2018 by the authors. Licensee MDPI, Basel, Switzerland.
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).
Coalition of Oct4A and β1 integrins in facilitating metastasis in ovarian cancer
- Samardzija, Chantel, Luwor, Rodney, Quinn, Michael, Kannourakis, George, Findlay, Jock, Ahmed, Nuzhat
- Authors: Samardzija, Chantel , Luwor, Rodney , Quinn, Michael , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2016
- Type: Text , Journal article
- Relation: BMC Cancer Vol. 16, no. 1 (2016), p. 1-16
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- Description: Background: Ovarian cancer is a metastatic disease and one of the leading causes of gynaecology malignancy-related deaths in women. Cancer stem cells (CSCs) are key contributors of cancer metastasis and relapse. Integrins are a family of cell surface receptors which allow interactions between cells and their surrounding microenvironment and play a fundamental role in promoting metastasis. This study investigates the molecular mechanism which associates CSCs and integrins in ovarian cancer metastasis. Methods: The expression of Oct4A in high-grade serous ovarian tumors and normal ovaries was determined by immunofluorescence analysis. The functional role of Oct4A was evaluated by generating stable knockdown (KD) of Oct4A clones in an established ovarian cancer cell line HEY using shRNA-mediated silencing. The expression of integrins in cell lines was evaluated by flow cytometry. Spheroid forming ability, adhesion and the activities of matrix metalloproteinases 9/2 (MMP-9/2) was measured by in vitro functional assays and gelatin zymography. These observations were further validated in in vivo mouse models using Balb/c nu/nu mice. Results: We report significantly elevated expression of Oct4A in high-grade serous ovarian tumors compared to normal ovarian tissues. The expression of Oct4A in ovarian cancer cell lines correlated with their CSC-related sphere forming abilities. The suppression of Oct4A in HEY cells resulted in a significant diminution of integrin β1 expression and associated α5 and α2 subunits compared to vector control cells. This was associated with a reduced adhesive ability on collagen and fibronectin and decreased secretion of pro-MMP2 in Oct4A KD cells compared to vector control cells. In vivo, Oct4A knock down (KD) cells produced tumors which were significantly smaller in size and weight compared to tumors derived from vector control cells. Immunohistochemical analyses of Oct4A KD tumor xenografts demonstrated a significant loss of cytokeratin 7 (CK7), Glut-1 as well as CD34 and CD31 compared to vector control cell-derived xenografts. Conclusion: The expression of Oct4A may be crucial to promote and sustain integrin-mediated extracellular matrix (ECM) remodeling requisite for tumor metastasis in ovarian cancer patients. © 2016 The Author(s).
- Authors: Samardzija, Chantel , Luwor, Rodney , Quinn, Michael , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2016
- Type: Text , Journal article
- Relation: BMC Cancer Vol. 16, no. 1 (2016), p. 1-16
- Full Text:
- Reviewed:
- Description: Background: Ovarian cancer is a metastatic disease and one of the leading causes of gynaecology malignancy-related deaths in women. Cancer stem cells (CSCs) are key contributors of cancer metastasis and relapse. Integrins are a family of cell surface receptors which allow interactions between cells and their surrounding microenvironment and play a fundamental role in promoting metastasis. This study investigates the molecular mechanism which associates CSCs and integrins in ovarian cancer metastasis. Methods: The expression of Oct4A in high-grade serous ovarian tumors and normal ovaries was determined by immunofluorescence analysis. The functional role of Oct4A was evaluated by generating stable knockdown (KD) of Oct4A clones in an established ovarian cancer cell line HEY using shRNA-mediated silencing. The expression of integrins in cell lines was evaluated by flow cytometry. Spheroid forming ability, adhesion and the activities of matrix metalloproteinases 9/2 (MMP-9/2) was measured by in vitro functional assays and gelatin zymography. These observations were further validated in in vivo mouse models using Balb/c nu/nu mice. Results: We report significantly elevated expression of Oct4A in high-grade serous ovarian tumors compared to normal ovarian tissues. The expression of Oct4A in ovarian cancer cell lines correlated with their CSC-related sphere forming abilities. The suppression of Oct4A in HEY cells resulted in a significant diminution of integrin β1 expression and associated α5 and α2 subunits compared to vector control cells. This was associated with a reduced adhesive ability on collagen and fibronectin and decreased secretion of pro-MMP2 in Oct4A KD cells compared to vector control cells. In vivo, Oct4A knock down (KD) cells produced tumors which were significantly smaller in size and weight compared to tumors derived from vector control cells. Immunohistochemical analyses of Oct4A KD tumor xenografts demonstrated a significant loss of cytokeratin 7 (CK7), Glut-1 as well as CD34 and CD31 compared to vector control cell-derived xenografts. Conclusion: The expression of Oct4A may be crucial to promote and sustain integrin-mediated extracellular matrix (ECM) remodeling requisite for tumor metastasis in ovarian cancer patients. © 2016 The Author(s).
Paclitaxel-induced Src activation is inhibited by dasatinib treatment, independently of cancer stem cell properties, in a mouse model of ovarian cancer
- Kadife, Elif, Chan, Emily, Luwor, Rodney, Kannourakis, George, Findlay, Jock, Ahmed, Nuzhat
- Authors: Kadife, Elif , Chan, Emily , Luwor, Rodney , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2019
- Type: Text , Journal article
- Relation: Cancers Vol. 11, no. 2 (2019), p. 1-24
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- Description: Approximately seventy percent of ovarian cancer patients succumb to the disease within the first 5 years of diagnosis, even after successful surgery and effective chemotherapy treatment. A small subset of chemotherapy resistant cancer stem cells (CSCs) cause relapse of ovarian cancers. This study investigated the association between paclitaxel-mediated Src activation (p-Src) and CSC populations in driving ovarian cancer progression. We demonstrate that patients with high-stage serous ovarian carcinomas have significantly elevated levels of p-Src, compared to patient with low-stage and benign ovarian tumours. Additionally, p-Src was significantly enhanced in ascites-derived tumour cells obtained from recurrent patients, compared to chemonaïve patients. Paclitaxel treatment increased Src activation in ovarian cancer cells, causing enrichment of CSC marker expression in the surviving cells in vitro and in xenografts of nude mice. Dasatinib in combination with paclitaxel significantly suppressed p-Src in ovarian cancer cell lines and xenografts but had no effect on the expression of CSC markers. However, combination of paclitaxel and Dasatinib showed lower trend in invasion in liver and pancreas, compared to paclitaxel-only treatment. The tumours treated with combination therapy also had significantly lower infiltration of mononuclear cells. Robust recurrent tumour growth was observed in all mice groups after termination of treatments. The above results suggest that Dasatinib-mediated inhibition of p-Src may not be crucial for paclitaxel-induced CSC-mediated recurrence in ovarian cancer.
- Authors: Kadife, Elif , Chan, Emily , Luwor, Rodney , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2019
- Type: Text , Journal article
- Relation: Cancers Vol. 11, no. 2 (2019), p. 1-24
- Full Text:
- Reviewed:
- Description: Approximately seventy percent of ovarian cancer patients succumb to the disease within the first 5 years of diagnosis, even after successful surgery and effective chemotherapy treatment. A small subset of chemotherapy resistant cancer stem cells (CSCs) cause relapse of ovarian cancers. This study investigated the association between paclitaxel-mediated Src activation (p-Src) and CSC populations in driving ovarian cancer progression. We demonstrate that patients with high-stage serous ovarian carcinomas have significantly elevated levels of p-Src, compared to patient with low-stage and benign ovarian tumours. Additionally, p-Src was significantly enhanced in ascites-derived tumour cells obtained from recurrent patients, compared to chemonaïve patients. Paclitaxel treatment increased Src activation in ovarian cancer cells, causing enrichment of CSC marker expression in the surviving cells in vitro and in xenografts of nude mice. Dasatinib in combination with paclitaxel significantly suppressed p-Src in ovarian cancer cell lines and xenografts but had no effect on the expression of CSC markers. However, combination of paclitaxel and Dasatinib showed lower trend in invasion in liver and pancreas, compared to paclitaxel-only treatment. The tumours treated with combination therapy also had significantly lower infiltration of mononuclear cells. Robust recurrent tumour growth was observed in all mice groups after termination of treatments. The above results suggest that Dasatinib-mediated inhibition of p-Src may not be crucial for paclitaxel-induced CSC-mediated recurrence in ovarian cancer.
Unique proteome signature of post-chemotherapy ovarian cancer ascites-derived tumor cells
- Ahmed, Nuzhat, Greening, David, Samardzija, Chantel, Escalona, Ruth, Chen, Maoshan, Findlay, Jock, Kannourakis, George
- Authors: Ahmed, Nuzhat , Greening, David , Samardzija, Chantel , Escalona, Ruth , Chen, Maoshan , Findlay, Jock , Kannourakis, George
- Date: 2016
- Type: Text , Journal article
- Relation: Scientific Reports Vol. 6, no. (2016), p. 1-13
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- Description: Eighty % of ovarian cancer patients diagnosed at an advanced-stage have complete remission after initial surgery and chemotherapy. However, most patients die within <5 years due to episodes of recurrences resulting from the growth of residual chemoresistant cells. In an effort to identify mechanisms associated with chemoresistance and recurrence, we compared the expression of proteins in ascites-derived tumor cells isolated from advanced-stage ovarian cancer patients obtained at diagnosis (chemonaive, CN) and after chemotherapy treatments (chemoresistant/at recurrence, CR) by using in-depth, high-resolution label-free quantitative proteomic profiling. A total of 2,999 proteins were identified. Using a stringent selection criterion to define only significantly differentially expressed proteins, we report identification of 353 proteins. There were significant differences in proteins encoding for immune surveillance, DNA repair mechanisms, cytoskeleton rearrangement, cell-cell adhesion, cell cycle pathways, cellular transport, and proteins involved with glycine/proline/arginine synthesis in tumor cells isolated from CR relative to CN patients. Pathway analyses revealed enrichment of metabolic pathways, DNA repair mechanisms and energy metabolism pathways in CR tumor cells. In conclusion, this is the first proteomics study to comprehensively analyze ascites-derived tumor cells from CN and CR ovarian cancer patients.
- Authors: Ahmed, Nuzhat , Greening, David , Samardzija, Chantel , Escalona, Ruth , Chen, Maoshan , Findlay, Jock , Kannourakis, George
- Date: 2016
- Type: Text , Journal article
- Relation: Scientific Reports Vol. 6, no. (2016), p. 1-13
- Full Text:
- Reviewed:
- Description: Eighty % of ovarian cancer patients diagnosed at an advanced-stage have complete remission after initial surgery and chemotherapy. However, most patients die within <5 years due to episodes of recurrences resulting from the growth of residual chemoresistant cells. In an effort to identify mechanisms associated with chemoresistance and recurrence, we compared the expression of proteins in ascites-derived tumor cells isolated from advanced-stage ovarian cancer patients obtained at diagnosis (chemonaive, CN) and after chemotherapy treatments (chemoresistant/at recurrence, CR) by using in-depth, high-resolution label-free quantitative proteomic profiling. A total of 2,999 proteins were identified. Using a stringent selection criterion to define only significantly differentially expressed proteins, we report identification of 353 proteins. There were significant differences in proteins encoding for immune surveillance, DNA repair mechanisms, cytoskeleton rearrangement, cell-cell adhesion, cell cycle pathways, cellular transport, and proteins involved with glycine/proline/arginine synthesis in tumor cells isolated from CR relative to CN patients. Pathway analyses revealed enrichment of metabolic pathways, DNA repair mechanisms and energy metabolism pathways in CR tumor cells. In conclusion, this is the first proteomics study to comprehensively analyze ascites-derived tumor cells from CN and CR ovarian cancer patients.
Momelotinib decreased cancer stem cell associated tumor burden and prolonged disease-free remission period in a mouse model of human ovarian cancer
- Chan, Emily, Luwor, Rodney, Burns, Christopher, Kannourakis, George, Findlay, Jock, Ahmed, Nuzhat
- Authors: Chan, Emily , Luwor, Rodney , Burns, Christopher , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2018
- Type: Text , Journal article
- Relation: Oncotarget Vol. 9, no. 24 (2018), p. 16599-16618
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- Description: Despite a good initial response to front-line chemotherapy, majority of the ovarian cancer patients relapse with consecutive phases of recurrences; and nearly 60% die within 5 years due to the development of a chemoresistant disease. This study investigated whether inhibition of the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway by momelotinib is sufficient in suppressing tumor burden and prolonging the disease-free survival period in a mouse model of ovarian cancer. We demonstrate that paclitaxel treatment enhanced JAK2/STAT3 activation which resulted in the enrichment of cancer stem cell (CSC)- like phenotype in the surviving ovarian cancer cells in vitro and in in vivo mouse xenografts. Combined treatment with paclitaxel and momelotinib inhibited paclitaxelinduced JAK2/STAT3 activation and CSC-like development in mice xenografts, and consequently reduced the tumor burden significantly greater than that achieved by paclitaxel-treatment alone. However, robust recurrent tumor growth with enhanced JAK2/STAT3 activation and CSC-like phenotype was observed in all mice groups after termination of treatments, but was delayed significantly in the paclitaxel and momelotinib treated group compared to other treatment groups. Daily oral gavage of momelotinib after termination of paclitaxel treatment showed sustained inhibition of tumor growth and a prolonged disease-free survival period in 50% of the mice. The other 50% of mice that developed tumors with ongoing momelotinib treatment also showed significantly increased survival benefit and a smaller tumor burden. These preliminary findings may have a profound clinical impact in developing an effective momelotinib-based 'maintenance-therapy' in ovarian cancer patients' postchemotherapy treatment. © Chan et al.
- Authors: Chan, Emily , Luwor, Rodney , Burns, Christopher , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2018
- Type: Text , Journal article
- Relation: Oncotarget Vol. 9, no. 24 (2018), p. 16599-16618
- Full Text:
- Reviewed:
- Description: Despite a good initial response to front-line chemotherapy, majority of the ovarian cancer patients relapse with consecutive phases of recurrences; and nearly 60% die within 5 years due to the development of a chemoresistant disease. This study investigated whether inhibition of the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway by momelotinib is sufficient in suppressing tumor burden and prolonging the disease-free survival period in a mouse model of ovarian cancer. We demonstrate that paclitaxel treatment enhanced JAK2/STAT3 activation which resulted in the enrichment of cancer stem cell (CSC)- like phenotype in the surviving ovarian cancer cells in vitro and in in vivo mouse xenografts. Combined treatment with paclitaxel and momelotinib inhibited paclitaxelinduced JAK2/STAT3 activation and CSC-like development in mice xenografts, and consequently reduced the tumor burden significantly greater than that achieved by paclitaxel-treatment alone. However, robust recurrent tumor growth with enhanced JAK2/STAT3 activation and CSC-like phenotype was observed in all mice groups after termination of treatments, but was delayed significantly in the paclitaxel and momelotinib treated group compared to other treatment groups. Daily oral gavage of momelotinib after termination of paclitaxel treatment showed sustained inhibition of tumor growth and a prolonged disease-free survival period in 50% of the mice. The other 50% of mice that developed tumors with ongoing momelotinib treatment also showed significantly increased survival benefit and a smaller tumor burden. These preliminary findings may have a profound clinical impact in developing an effective momelotinib-based 'maintenance-therapy' in ovarian cancer patients' postchemotherapy treatment. © Chan et al.
Expression of TIMPs and MMPs in ovarian tumors, ascites, ascites-derived cells, and cancer cell lines : characteristic modulatory response before and after chemotherapy treatment
- Escalona, Ruth, Kannourakis, George, Findlay, Jock, Ahmed, Nuzhat
- Authors: Escalona, Ruth , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2022
- Type: Text , Journal article
- Relation: Frontiers in Oncology Vol. 11, no. (2022), p.
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- Description: Background: The tissue inhibitors of metalloproteinase (TIMPs) and their associated metalloproteinase (MMPs) are essential regulators of tissue homeostasis and are essential for cancer progression. This study analyzed the expression of TIMP-1,-2,-3 and the associated MMPs (MMP-2,-9,-11,-14) in different Stages, Grades and World Health Organization (WHO) classifications of serous ovarian tumors, ascites, ascites-derived cells from chemo-naïve (CN) and relapsed (CR) patients, and in ovarian cancer cell lines. The status of TIMPs and associated MMPs in response to chemotherapy treatment was assessed in cancer cell lines; TCGA data was interrogated to gauge TIMPs and associated MMPs as prognostic and platinum-response indicators. Methods: The levels of TIMP-1, -2 and -3 were assessed by immunohistochemistry. The mRNA expression of TIMPs and MMPs was quantified by real time PCR (qRT-PCR). The chemosensitivity (IC50 values) to Cisplatin or Paclitaxel in cell lines was evaluated by MTT assay. The levels of TIMPs in ascites and cell lysates were analyzed by an ELISA assay. Results: The expression of TIMP-2 was significantly upregulated in Type 2 compared to Type 1 tumors and normal/benign ovarian tissues. TIMP-3 expression was significantly enhanced in Stage III, Grade 3 and Type 2 tumors compared to normal/benign ovarian tissues. The mRNA expression of MMP-9,-11 and -14 was significantly upregulated in Stage IV compared to normal/benign ovarian tissues. The expression of TIMP-1 was highest, followed by TIMP-2 and then TIMP-3 in CN ascites. At the cellular level, TIMP-2 mRNA expression was significantly higher in CN compared to CR epithelial cells in patients. The expression of TIMP-1 and -2, MMPs and cancer stem cells (CSCs) were upregulated in response to chemotherapy treatments in cancer cell lines. Interrogation of the TCGA dataset suggests shifts in platinum responses in patients consistent with genetic alterations in TIMP-2, -3 and MMP-2, -11 genes in tumors; and decreased overall survival (OS) and progression-free survival (PFS) in patients with altered MMP-14 genes. Conclusions: TIMPs and related MMPs are differentially expressed in serous ovarian tumors, ascites, ascites-derived cells and ovarian cancer cell lines. Chemotherapy treatment modulates expression of TIMPs and MMPs in association with increased expression of genes related to cancer stem cells. Copyright © 2022 Escalona, Kannourakis, Findlay and Ahmed.
- Authors: Escalona, Ruth , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2022
- Type: Text , Journal article
- Relation: Frontiers in Oncology Vol. 11, no. (2022), p.
- Full Text:
- Reviewed:
- Description: Background: The tissue inhibitors of metalloproteinase (TIMPs) and their associated metalloproteinase (MMPs) are essential regulators of tissue homeostasis and are essential for cancer progression. This study analyzed the expression of TIMP-1,-2,-3 and the associated MMPs (MMP-2,-9,-11,-14) in different Stages, Grades and World Health Organization (WHO) classifications of serous ovarian tumors, ascites, ascites-derived cells from chemo-naïve (CN) and relapsed (CR) patients, and in ovarian cancer cell lines. The status of TIMPs and associated MMPs in response to chemotherapy treatment was assessed in cancer cell lines; TCGA data was interrogated to gauge TIMPs and associated MMPs as prognostic and platinum-response indicators. Methods: The levels of TIMP-1, -2 and -3 were assessed by immunohistochemistry. The mRNA expression of TIMPs and MMPs was quantified by real time PCR (qRT-PCR). The chemosensitivity (IC50 values) to Cisplatin or Paclitaxel in cell lines was evaluated by MTT assay. The levels of TIMPs in ascites and cell lysates were analyzed by an ELISA assay. Results: The expression of TIMP-2 was significantly upregulated in Type 2 compared to Type 1 tumors and normal/benign ovarian tissues. TIMP-3 expression was significantly enhanced in Stage III, Grade 3 and Type 2 tumors compared to normal/benign ovarian tissues. The mRNA expression of MMP-9,-11 and -14 was significantly upregulated in Stage IV compared to normal/benign ovarian tissues. The expression of TIMP-1 was highest, followed by TIMP-2 and then TIMP-3 in CN ascites. At the cellular level, TIMP-2 mRNA expression was significantly higher in CN compared to CR epithelial cells in patients. The expression of TIMP-1 and -2, MMPs and cancer stem cells (CSCs) were upregulated in response to chemotherapy treatments in cancer cell lines. Interrogation of the TCGA dataset suggests shifts in platinum responses in patients consistent with genetic alterations in TIMP-2, -3 and MMP-2, -11 genes in tumors; and decreased overall survival (OS) and progression-free survival (PFS) in patients with altered MMP-14 genes. Conclusions: TIMPs and related MMPs are differentially expressed in serous ovarian tumors, ascites, ascites-derived cells and ovarian cancer cell lines. Chemotherapy treatment modulates expression of TIMPs and MMPs in association with increased expression of genes related to cancer stem cells. Copyright © 2022 Escalona, Kannourakis, Findlay and Ahmed.
Knock down of TIMP-2 by siRNA and CRISPR/Cas9 mediates diverse cellular reprogramming of metastasis and chemosensitivity in ovarian cancer
- Escalona, Ruth, Chu, Simon, Kadife, Elif, Kelly, Jason, Kannourakis, George, Findlay, Jock, Ahmed, Nuzhat
- Authors: Escalona, Ruth , Chu, Simon , Kadife, Elif , Kelly, Jason , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2022
- Type: Text , Journal article
- Relation: Cancer Cell International Vol. 22, no. 1 (2022), p.
- Full Text:
- Reviewed:
- Description: Background: The endogenous tissue inhibitor of metalloproteinase-2 (TIMP-2), through its homeostatic action on certain metalloproteinases, plays a vital role in remodelling extracellular matrix (ECM) to facilitate cancer progression. This study investigated the role of TIMP-2 in an ovarian cancer cell line in which the expression of TIMP-2 was reduced by either siRNA or CRISPR/Cas9. Methods: OVCAR5 cells were transiently and stably transfected with either single or pooled TIMP-2 siRNAs (T2-KD cells) or by CRISPR/Cas9 under the influence of two distinct guide RNAs (gRNA1 and gRNA2 cell lines). The expression of different genes was analysed at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence (IF) and western blot. Proliferation of cells was investigated by 5-Ethynyl-2′-deoxyuridine (EdU) assay or staining with Ki67. Cell migration/invasion was determined by xCELLigence. Cell growth in vitro was determined by 3D spheroid cultures and in vivo by a mouse xenograft model. Results: Approximately 70–90% knock down of TIMP-2 expression were confirmed in T2-KD, gRNA1 and gRNA2 OVCAR5 ovarian cancer cells at the protein level. T2-KD, gRNA1 and gRNA2 cells exhibited a significant downregulation of MMP-2 expression, but concurrently a significant upregulation in the expression of membrane bound MMP-14 compared to control and parental cells. Enhanced proliferation and invasion were exhibited in all TIMP-2 knocked down cells but differences in sensitivity to paclitaxel (PTX) treatment were observed, with T2-KD cells and gRNA2 cell line being sensitive, while the gRNA1 cell line was resistant to PTX treatment. In addition, significant differences in the growth of gRNA1 and gRNA2 cell lines were observed in in vitro 3D cultures as well as in an in vivo mouse xenograft model. Conclusions: Our results suggest that the inhibition of TIMP-2 by siRNA and CRISPR/Cas-9 modulate the expression of MMP-2 and MMP-14 and reprogram ovarian cancer cells to facilitate proliferation and invasion. Distinct disparities in in vitro chemosensitivity and growth in 3D culture, and differences in tumour burden and invasion to proximal organs in a mouse model imply that selective suppression of TIMP-2 expression by siRNA or CRISPR/Cas-9 alters important aspects of metastasis and chemosensitivity in ovarian cancer. © 2022, The Author(s).
- Authors: Escalona, Ruth , Chu, Simon , Kadife, Elif , Kelly, Jason , Kannourakis, George , Findlay, Jock , Ahmed, Nuzhat
- Date: 2022
- Type: Text , Journal article
- Relation: Cancer Cell International Vol. 22, no. 1 (2022), p.
- Full Text:
- Reviewed:
- Description: Background: The endogenous tissue inhibitor of metalloproteinase-2 (TIMP-2), through its homeostatic action on certain metalloproteinases, plays a vital role in remodelling extracellular matrix (ECM) to facilitate cancer progression. This study investigated the role of TIMP-2 in an ovarian cancer cell line in which the expression of TIMP-2 was reduced by either siRNA or CRISPR/Cas9. Methods: OVCAR5 cells were transiently and stably transfected with either single or pooled TIMP-2 siRNAs (T2-KD cells) or by CRISPR/Cas9 under the influence of two distinct guide RNAs (gRNA1 and gRNA2 cell lines). The expression of different genes was analysed at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence (IF) and western blot. Proliferation of cells was investigated by 5-Ethynyl-2′-deoxyuridine (EdU) assay or staining with Ki67. Cell migration/invasion was determined by xCELLigence. Cell growth in vitro was determined by 3D spheroid cultures and in vivo by a mouse xenograft model. Results: Approximately 70–90% knock down of TIMP-2 expression were confirmed in T2-KD, gRNA1 and gRNA2 OVCAR5 ovarian cancer cells at the protein level. T2-KD, gRNA1 and gRNA2 cells exhibited a significant downregulation of MMP-2 expression, but concurrently a significant upregulation in the expression of membrane bound MMP-14 compared to control and parental cells. Enhanced proliferation and invasion were exhibited in all TIMP-2 knocked down cells but differences in sensitivity to paclitaxel (PTX) treatment were observed, with T2-KD cells and gRNA2 cell line being sensitive, while the gRNA1 cell line was resistant to PTX treatment. In addition, significant differences in the growth of gRNA1 and gRNA2 cell lines were observed in in vitro 3D cultures as well as in an in vivo mouse xenograft model. Conclusions: Our results suggest that the inhibition of TIMP-2 by siRNA and CRISPR/Cas-9 modulate the expression of MMP-2 and MMP-14 and reprogram ovarian cancer cells to facilitate proliferation and invasion. Distinct disparities in in vitro chemosensitivity and growth in 3D culture, and differences in tumour burden and invasion to proximal organs in a mouse model imply that selective suppression of TIMP-2 expression by siRNA or CRISPR/Cas-9 alters important aspects of metastasis and chemosensitivity in ovarian cancer. © 2022, The Author(s).
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