GGNBP2 to suppress tumor growth and cancer stem cell load of triple negative breast cancer by controlling STAT3 pathway.

e23003 Background: Gametogenetin binding protein 2 (GGNBP2) is encoded in human chromosome 17q12-q23, a region known as a breast and ovarian cancer susceptibility locus. GGNBP2 has a single C2H2 zinc finger and a consensus LxxLL nuclear receptor (NR) binding motif. We have reported that GGNBP2 suppresses ERα-positive breast tumorigenesis by acting as a nuclear receptor co-repressor to restrain ERα activity. However, the detailed molecular mechanisms of GGNBP2 and its role in triple negative breast cancer (TNBC) remain largely unclear. Methods: A human breast cancer tissue array containing 138 human breast tumor tissues were utilized to examine GGNBP2 expression in breast cancer samples by IHC. To address the potential anti-breast tumor activity of GGNBP2 in vitro, we expressed exogenous GGNBP2 in TNBC cells, including MDA-MB-231 and Cal51 cell lines. Cell proliferation and cell cycle were assessed by cell growth curve/EdU assays and flow cytometry after propidium Iodide staining. Apoptosis was determined by flow cytometry after annexin V staining, by caspase 3/7 and caspase 9 activity assays. Cancer stem cell properties were determined by expression of CD44/CD24/ALDH1 markers. The levels of phosphorylated STAT3 and total STAT3 were determined by western blot. Quantitative PCR and Western blot were carried out to evaluate the effects of GGNBP2 overexpression on STAT3 target genes, CCND1, Mcl-1, survivin, bax and bim expression. Results: GGNBP2 expression is down-regulated in TNBC cells and patient tumors. GGNBP2 down-regulated expression is associated with poor patient survival. Overexpression of GGNBP2 significanly induces cell cycle G0/G1 phase arrest and apoptosis in TNBC cell lines. Expression of cancer stem cell markers also decreased in GGNBP2-overexpressed TNBC cells. GGNBP2 reduces the expression levels of CCND1, Mcl-1 and survivin, promotes the expression levels of bax and bim proteins. Importantly, overexpression of GGNBP2 inhibits STAT3 phosphorylation and STAT3 downstream target gene expression, including CCND1, Mcl-1 and survivin. Conclusions: GGNBP2 serves as a critical nuclear negative regulator of STAT3-mediated gene expression and tumorigenesis in TNBC.

Download Full-text

EP300 Knockdown Abrogates Cancer Stem Cell Phenotype, Tumor Growth and Metastasis in Triple Negative Breast Cancer

10.21203/rs.3.rs-25458/v1 ◽

2020 ◽

Author(s):

Alexander Ring ◽

Pushpinder Kaur ◽

Julie E. Lang

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Stem Cell ◽

Cancer Stem Cell ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Side Population ◽

Tumor Growth And Metastasis

Abstract Background:Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with basal features, lacking the expression of receptors targeted successfully in other breast cancer subtypes. Treatment response to adjuvant and neoadjuvant chemotherapy is often short-lived and metastatic spread occurs at higher rates than other subtypes within the first five years after diagnosis. TNBCs exhibit stem cell features and are enriched for cancer stem cell (CSC) populations. E1A Binding Protein P300(EP300) is a large protein with multiple cellular functions, including as an effector in stem cell biology.Methods: We used a genetic knockdown (KD) model of EP300 in TNBC cell lines to investigate the effect on CSC phenotype, tumor growth and metastasis. Side population assay and tumorsphere suspension culture were used in vitro. Xenograft mouse models were used for in vivo studies. We performedin silico analysis of publicly available gene expression data sets to investigate CSC gene expression and molecular pathways as well as survival outcomes associated with EP300 expression in patients with TNBC and basal-like BC.Results: EP300 KD abolishedthe CSC phenotype by reducing ABCG2 expression, side population cells andtumorsphere formation capacityin vitro as well as tumor formation in a xenograft mouse model in vivo. Metastatic capacity was markedly reduced in EP300 KD cells in vivo, with no detection of circulating tumor cells.TCGA data analysis demonstrated that genes positively correlated with EP300 expression in TNBC and basal-like BC were associated with CSC biology. Survival analysis demonstrated that EP300 expression predicts poor recurrence free survival in TNBC and basal BC. Conclusion:We report a novel oncogenic role for EP300 in driving CSC phenotyperepresentinga potential target to address tumor initiation and metastatic spread in TNBC and basal-like BC. EP300 might serve as a prognostic marker and potential therapeutic target in TNBC.

Download Full-text

EP300 Knockdown Reduces Cancer Stem Cell Phenotype, Tumor Growth and Metastasis in Triple Negative Breast Cancer

10.21203/rs.3.rs-25458/v4 ◽

2020 ◽

Author(s):

Alexander Ring ◽

Pushpinder Kaur ◽

Julie E. Lang

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Stem Cell ◽

Cancer Stem Cell ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Side Population ◽

Tumor Growth And Metastasis

Abstract Background: Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with basal features, lacking the expression of receptors targeted successfully in other breast cancer subtypes. Treatment response to adjuvant and neoadjuvant chemotherapy is often short-lived and metastatic spread occurs at higher rates than other subtypes within the first five years after diagnosis. TNBCs exhibit stem cell features and are enriched for cancer stem cell (CSC) populations. E1A Binding Protein P300 (EP300) is a large protein with multiple cellular functions, including as an effector in stem cell biology.Methods: We used a genetic knockdown (KD) model of EP300 in TNBC cell lines to investigate the effect on CSC phenotype, tumor growth and metastasis. Side population assay and tumorsphere suspension culture were used in vitro. Xenograft mouse models were used for in vivo studies. We performed in silico analysis of publicly available gene expression data sets to investigate CSC gene expression and molecular pathways as well as survival outcomes associated with EP300 expression in patients with TNBC and basal-like BC.Results: EP300 KD abolished the CSC phenotype by reducing ABCG2 expression, side population cells and tumorsphere formation capacity in vitro as well as tumor formation in a xenograft mouse model in vivo. Metastatic capacity was markedly reduced in EP300 KD cells in vivo, with no detection of circulating tumor cells. TCGA data analysis demonstrated that genes positively correlated with EP300 expression in TNBC and basal-like BC were associated with CSC biology. Survival analysis demonstrated that EP300 expression predicts poor recurrence free survival in TNBC and basal BC. Conclusion: We report a novel oncogenic role for EP300 in driving CSC phenotype representing a potential target to address tumor initiation and metastatic spread in TNBC and basal-like BC. EP300 might serve as a prognostic marker and potential therapeutic target in TNBC.

Download Full-text

EP300 Knockdown Reduces Cancer Stem Cell Phenotype, Tumor Growth and Metastasis in Triple Negative Breast Cancer

10.21203/rs.3.rs-25458/v2 ◽

2020 ◽

Author(s):

Alexander Ring ◽

Pushpinder Kaur ◽

Julie E. Lang

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Stem Cell ◽

Cancer Stem Cell ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Side Population ◽

Tumor Growth And Metastasis

Abstract Background: Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with basal features, lacking the expression of receptors targeted successfully in other breast cancer subtypes. Treatment response to adjuvant and neoadjuvant chemotherapy is often short-lived and metastatic spread occurs at higher rates than other subtypes within the first five years after diagnosis. TNBCs exhibit stem cell features and are enriched for cancer stem cell (CSC) populations. E1A Binding Protein P300 (EP300) is a large protein with multiple cellular functions, including as an effector in stem cell biology.Methods: We used a genetic knockdown (KD) model of EP300 in TNBC cell lines to investigate the effect on CSC phenotype, tumor growth and metastasis. Side population assay and tumorsphere suspension culture were used in vitro. Xenograft mouse models were used for in vivo studies. We performed in silico analysis of publicly available gene expression data sets to investigate CSC gene expression and molecular pathways as well as survival outcomes associated with EP300 expression in patients with TNBC and basal-like BC.Results: EP300 KD abolished the CSC phenotype by reducing ABCG2 expression, side population cells and tumorsphere formation capacity in vitro as well as tumor formation in a xenograft mouse model in vivo. Metastatic capacity was markedly reduced in EP300 KD cells in vivo, with no detection of circulating tumor cells. TCGA data analysis demonstrated that genes positively correlated with EP300 expression in TNBC and basal-like BC were associated with CSC biology. Survival analysis demonstrated that EP300 expression predicts poor recurrence free survival in TNBC and basal BC. Conclusion: We report a novel oncogenic role for EP300 in driving CSC phenotype representing a potential target to address tumor initiation and metastatic spread in TNBC and basal-like BC. EP300 might serve as a prognostic marker and potential therapeutic target in TNBC.

Download Full-text

EP300 Knockdown Reduces Cancer Stem Cell Phenotype, Tumor Growth and Metastasis in Triple Negative Breast Cancer

10.21203/rs.3.rs-25458/v3 ◽

2020 ◽

Author(s):

Alexander Ring ◽

Pushpinder Kaur ◽

Julie E. Lang

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Stem Cell ◽

Cancer Stem Cell ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Side Population ◽

Tumor Growth And Metastasis

Abstract Background: Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with basal features, lacking the expression of receptors targeted successfully in other breast cancer subtypes. Treatment response to adjuvant and neoadjuvant chemotherapy is often short-lived and metastatic spread occurs at higher rates than other subtypes within the first five years after diagnosis. TNBCs exhibit stem cell features and are enriched for cancer stem cell (CSC) populations. E1A Binding Protein P300 (EP300) is a large protein with multiple cellular functions, including as an effector in stem cell biology.Methods: We used a genetic knockdown (KD) model of EP300 in TNBC cell lines to investigate the effect on CSC phenotype, tumor growth and metastasis. Side population assay and tumorsphere suspension culture were used in vitro. Xenograft mouse models were used for in vivo studies. We performed in silico analysis of publicly available gene expression data sets to investigate CSC gene expression and molecular pathways as well as survival outcomes associated with EP300 expression in patients with TNBC and basal-like BC.Results: EP300 KD abolished the CSC phenotype by reducing ABCG2 expression, side population cells and tumorsphere formation capacity in vitro as well as tumor formation in a xenograft mouse model in vivo. Metastatic capacity was markedly reduced in EP300 KD cells in vivo, with no detection of circulating tumor cells. TCGA data analysis demonstrated that genes positively correlated with EP300 expression in TNBC and basal-like BC were associated with CSC biology. Survival analysis demonstrated that EP300 expression predicts poor recurrence free survival in TNBC and basal BC.Conclusion: We report a novel oncogenic role for EP300 in driving CSC phenotype representing a potential target to address tumor initiation and metastatic spread in TNBC and basal-like BC. EP300 might serve as a prognostic marker and potential therapeutic target in TNBC.

Download Full-text

EP300 knockdown reduces cancer stem cell phenotype, tumor growth and metastasis in triple negative breast cancer

BMC Cancer ◽

10.1186/s12885-020-07573-y ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Alexander Ring ◽

Pushpinder Kaur ◽

Julie E. Lang

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Stem Cell ◽

Cancer Stem Cell ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Side Population ◽

Tumor Growth And Metastasis

Abstract Background Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with basal features, lacking the expression of receptors targeted successfully in other breast cancer subtypes. Treatment response to adjuvant and neoadjuvant chemotherapy is often short-lived and metastatic spread occurs at higher rates than other subtypes within the first five years after diagnosis. TNBCs exhibit stem cell features and are enriched for cancer stem cell (CSC) populations. E1A Binding Protein P300 (EP300) is a large protein with multiple cellular functions, including as an effector in stem cell biology. Methods We used a genetic knockdown (KD) model of EP300 in TNBC cell lines to investigate the effect on CSC phenotype, tumor growth and metastasis. Side population assay and tumorsphere suspension culture were used in vitro. Xenograft mouse models were used for in vivo studies. We performed in silico analysis of publicly available gene expression data sets to investigate CSC gene expression and molecular pathways as well as survival outcomes associated with EP300 expression in patients with TNBC and basal-like BC. Results EP300 KD abolished the CSC phenotype by reducing ABCG2 expression, side population cells and tumorsphere formation capacity in vitro as well as tumor formation in a xenograft mouse model in vivo. Metastatic capacity was markedly reduced in EP300 KD cells in vivo, with no detection of circulating tumor cells. TCGA data analysis demonstrated that genes positively correlated with EP300 expression in TNBC and basal-like BC were associated with CSC biology. Survival analysis demonstrated that EP300 expression predicts poor recurrence free survival in TNBC and basal BC. Conclusion We report a novel oncogenic role for EP300 in driving CSC phenotype representing a potential target to address tumor initiation and metastatic spread in TNBC and basal-like BC. EP300 might serve as a prognostic marker and potential therapeutic target in TNBC.

Download Full-text

Isolation and Establishment of a Highly Proliferative, Cancer Stem Cell-Like, and Naturally Immortalized Triple-Negative Breast Cancer Cell Line, KAIMRC2

Cells ◽

10.3390/cells10061303 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1303

Author(s):

Rizwan Ali ◽

Hajar Al Zahrani ◽

Tlili Barhoumi ◽

Alshaimaa Alhallaj ◽

Abdullah Mashhour ◽

...

Keyword(s):

Breast Cancer ◽

Stem Cell ◽

Cancer Stem Cell ◽

Cell Line ◽

Cancer Cell ◽

Triple Negative Breast Cancer ◽

Breast Cancer Cell Line ◽

Triple Negative ◽

Cancer Cell Line

In vitro studies of a disease are key to any in vivo investigation in understanding the disease and developing new therapy regimens. Immortalized cancer cell lines are the best and easiest model for studying cancer in vitro. Here, we report the establishment of a naturally immortalized highly tumorigenic and triple-negative breast cancer cell line, KAIMRC2. This cell line is derived from a Saudi Arabian female breast cancer patient with invasive ductal carcinoma. Immunocytochemistry showed a significant ratio of the KAIMRC2 cells’ expressing key breast epithelial and cancer stem cells (CSCs) markers, including CD47, CD133, CD49f, CD44, and ALDH-1A1. Gene and protein expression analysis showed overexpression of ABC transporter and AKT-PI3Kinase as well as JAK/STAT signaling pathways. In contrast, the absence of the tumor suppressor genes p53 and p73 may explain their high proliferative index. The mice model also confirmed the tumorigenic potential of the KAIMRC2 cell line, and drug tolerance studies revealed few very potent candidates. Our results confirmed an aggressive phenotype with metastatic potential and cancer stem cell-like characteristics of the KAIMR2 cell line. Furthermore, we have also presented potent small molecule inhibitors, especially Ryuvidine, that can be further developed, alone or in synergy with other potent inhibitors, to target multiple cancer-related pathways.

Download Full-text