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SOCS3-mediated regulation of inflammatory cytokines in PTEN and p53 inactivated triple negative breast cancer model.

Kim G, Ouzounova M, Quraishi AA, Davis A, Tawakkol N, Clouthier SG, Malik F, Paulson AK, D'Angelo RC, Korkaya S, Baker TL, Esen ES, Prat A, Liu S, Kleer CG, Thomas DG, Wicha MS, Korkaya H - Oncogene (2014)

Bottom Line: Constitutive activation of this loop in transformed cells was dependent on proteolytic degradation of suppressor of cytokine signaling 3 (SOCS3) resulting in low levels of this protein in basal/claudin-low cell lines and primary tumors.In transformed cells, enforced expression of SOCS3 or interfering with IL6 pathway via IL6R blockade inhibited tumor growth and metastasis in mouse xenograft models.Furthermore, circulating tumor cells were significantly reduced in tumor-bearing animals when treated with anti-IL6R antibodies.

View Article: PubMed Central - PubMed

Affiliation: 1] Comprehensive Cancer Center, Department of Internal medicine, University of Michigan, Ann Arbor, MI, USA [2] Department of Pathology, CHA Bundang Medical Center, CHA University, Seongnam 463-712, Gyeonggi, Republic of Korea.

ABSTRACT
Somatic mutations or deletions of TP53 and PTEN in ductal carcinoma in situ lesions have been implicated in progression to invasive ductal carcinomas. A recent molecular and mutational analysis of breast cancers revealed that inactivation of tumor suppressors, p53 and PTEN, are strongly associated with triple negative breast cancer. In addition, these tumor suppressors have important roles in regulating self-renewal in normal and malignant stem cells. To investigate their role in breast carcinogenesis, we knocked down these genes in human mammary cells and in non-transformed MCF10A cells. p53 and PTEN knockdown synergized to activate pro-inflammatory interleukin-6 (IL6)/Stat3/nuclear factor κB signaling. This resulted in generation of highly metastatic epithelial-to-mesenchymal transition-like cancer stem cells resulting in tumors whose gene expression profile mimicked that found in basal/claudin-low molecular subtype within the triple negative breast tumors. Constitutive activation of this loop in transformed cells was dependent on proteolytic degradation of suppressor of cytokine signaling 3 (SOCS3) resulting in low levels of this protein in basal/claudin-low cell lines and primary tumors. In non-transformed cells, transient activation of the IL6 inflammatory loop induced SOCS3 expression leading to pathway inactivation. In transformed cells, enforced expression of SOCS3 or interfering with IL6 pathway via IL6R blockade inhibited tumor growth and metastasis in mouse xenograft models. Furthermore, circulating tumor cells were significantly reduced in tumor-bearing animals when treated with anti-IL6R antibodies. These studies uncover important connections between inflammation and carcinogenesis and suggest that blocking pro-inflammatory cytokines may be utilized as an attractive strategy to target triple negative breast tumors, which currently lacks molecularly targeted therapies.

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p53 and PTEN knockdown in mammary epithelial cells activates inflammatory Stat3/NF-κB pathway expanding stem cell population(a) Western blot analyses of MCF10A, p53 (p53−), PTEN (PTEN−) or combined p53 and PTEN knockdown cells (p53−PTEN−) show expressions of p53, PTEN and EMT markers, Vimentin, E-cadherin as well as the activations of Stat3/NF-κB and Akt/Wnt/b-catenin pathways. (b) Sphere forming assay in control HNMEC and MCF10A cells compared to p53−, PTEN− or p53−PTEN− cells; scale bar, 100μm. (c) Number of spheres formed per 10,000 cells plated. (d) Flow cytometry analysis of p53− and PTEN− cells with CD44/CD24 and EpCAM/CD49f markers. (e, f) Quantification of CD44+CD24− cells and EpCAM/CD49f subpopulations were analyzed by flow cytometry. Means ± SD (n=3),*p≤0.05, ** p≤0.005.
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Figure 1: p53 and PTEN knockdown in mammary epithelial cells activates inflammatory Stat3/NF-κB pathway expanding stem cell population(a) Western blot analyses of MCF10A, p53 (p53−), PTEN (PTEN−) or combined p53 and PTEN knockdown cells (p53−PTEN−) show expressions of p53, PTEN and EMT markers, Vimentin, E-cadherin as well as the activations of Stat3/NF-κB and Akt/Wnt/b-catenin pathways. (b) Sphere forming assay in control HNMEC and MCF10A cells compared to p53−, PTEN− or p53−PTEN− cells; scale bar, 100μm. (c) Number of spheres formed per 10,000 cells plated. (d) Flow cytometry analysis of p53− and PTEN− cells with CD44/CD24 and EpCAM/CD49f markers. (e, f) Quantification of CD44+CD24− cells and EpCAM/CD49f subpopulations were analyzed by flow cytometry. Means ± SD (n=3),*p≤0.05, ** p≤0.005.

Mentions: In order to investigate the molecular changes induced by inactivation of tumor suppressors in breast cancer, we down regulated p53 (p53−) and PTEN (PTEN−) individually or in combination (p53−PTEN−) in normal human mammary epithelial cells (HNMEC) and immortalized MCF10A cells (Figures 1a and Supplementary 1a). Simultaneous knock down of p53 and PTEN in MCF10A cells results in activation of Akt/β-catenin and Stat3/NF-κB pathways as well as increased expression of Vimentin and decreased expression of E-cadherin (Figure 1a). To determine the functional relevance of pathway activation, we utilized the non-adherent sphere forming assay to assess mammary stem cell activity in vitro (7). p53 and PTEN knockdown in HNMECs and MCF10A cell line increased the sphere formation 2–10 fold compared to control or single gene deleted cells (Figure 1b and c). Utilizing the CSC markers, CD44+CD24− (8) as well as CD49f+EpCAM− which represents the mesenchymal CSC phenotype (9), we demonstrated that MCF10Ap53−PTEN− cells display a significant increase in the CD44+CD24− cell population compared to parental, p53− or PTEN− cells (Figures 1d and e). Although MCF10Ap53−PTEN− cells contained an increased EpCAM−CD49f+ population, these cells also displayed a distinct EpCAM−CD49f− population not found in parental MCF10A, MCF10A-p53− or MCF10A-PTEN− cells which are predominantly EpCAM+CD49f+ (Figures 1d and f).


SOCS3-mediated regulation of inflammatory cytokines in PTEN and p53 inactivated triple negative breast cancer model.

Kim G, Ouzounova M, Quraishi AA, Davis A, Tawakkol N, Clouthier SG, Malik F, Paulson AK, D'Angelo RC, Korkaya S, Baker TL, Esen ES, Prat A, Liu S, Kleer CG, Thomas DG, Wicha MS, Korkaya H - Oncogene (2014)

p53 and PTEN knockdown in mammary epithelial cells activates inflammatory Stat3/NF-κB pathway expanding stem cell population(a) Western blot analyses of MCF10A, p53 (p53−), PTEN (PTEN−) or combined p53 and PTEN knockdown cells (p53−PTEN−) show expressions of p53, PTEN and EMT markers, Vimentin, E-cadherin as well as the activations of Stat3/NF-κB and Akt/Wnt/b-catenin pathways. (b) Sphere forming assay in control HNMEC and MCF10A cells compared to p53−, PTEN− or p53−PTEN− cells; scale bar, 100μm. (c) Number of spheres formed per 10,000 cells plated. (d) Flow cytometry analysis of p53− and PTEN− cells with CD44/CD24 and EpCAM/CD49f markers. (e, f) Quantification of CD44+CD24− cells and EpCAM/CD49f subpopulations were analyzed by flow cytometry. Means ± SD (n=3),*p≤0.05, ** p≤0.005.
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Figure 1: p53 and PTEN knockdown in mammary epithelial cells activates inflammatory Stat3/NF-κB pathway expanding stem cell population(a) Western blot analyses of MCF10A, p53 (p53−), PTEN (PTEN−) or combined p53 and PTEN knockdown cells (p53−PTEN−) show expressions of p53, PTEN and EMT markers, Vimentin, E-cadherin as well as the activations of Stat3/NF-κB and Akt/Wnt/b-catenin pathways. (b) Sphere forming assay in control HNMEC and MCF10A cells compared to p53−, PTEN− or p53−PTEN− cells; scale bar, 100μm. (c) Number of spheres formed per 10,000 cells plated. (d) Flow cytometry analysis of p53− and PTEN− cells with CD44/CD24 and EpCAM/CD49f markers. (e, f) Quantification of CD44+CD24− cells and EpCAM/CD49f subpopulations were analyzed by flow cytometry. Means ± SD (n=3),*p≤0.05, ** p≤0.005.
Mentions: In order to investigate the molecular changes induced by inactivation of tumor suppressors in breast cancer, we down regulated p53 (p53−) and PTEN (PTEN−) individually or in combination (p53−PTEN−) in normal human mammary epithelial cells (HNMEC) and immortalized MCF10A cells (Figures 1a and Supplementary 1a). Simultaneous knock down of p53 and PTEN in MCF10A cells results in activation of Akt/β-catenin and Stat3/NF-κB pathways as well as increased expression of Vimentin and decreased expression of E-cadherin (Figure 1a). To determine the functional relevance of pathway activation, we utilized the non-adherent sphere forming assay to assess mammary stem cell activity in vitro (7). p53 and PTEN knockdown in HNMECs and MCF10A cell line increased the sphere formation 2–10 fold compared to control or single gene deleted cells (Figure 1b and c). Utilizing the CSC markers, CD44+CD24− (8) as well as CD49f+EpCAM− which represents the mesenchymal CSC phenotype (9), we demonstrated that MCF10Ap53−PTEN− cells display a significant increase in the CD44+CD24− cell population compared to parental, p53− or PTEN− cells (Figures 1d and e). Although MCF10Ap53−PTEN− cells contained an increased EpCAM−CD49f+ population, these cells also displayed a distinct EpCAM−CD49f− population not found in parental MCF10A, MCF10A-p53− or MCF10A-PTEN− cells which are predominantly EpCAM+CD49f+ (Figures 1d and f).

Bottom Line: Constitutive activation of this loop in transformed cells was dependent on proteolytic degradation of suppressor of cytokine signaling 3 (SOCS3) resulting in low levels of this protein in basal/claudin-low cell lines and primary tumors.In transformed cells, enforced expression of SOCS3 or interfering with IL6 pathway via IL6R blockade inhibited tumor growth and metastasis in mouse xenograft models.Furthermore, circulating tumor cells were significantly reduced in tumor-bearing animals when treated with anti-IL6R antibodies.

View Article: PubMed Central - PubMed

Affiliation: 1] Comprehensive Cancer Center, Department of Internal medicine, University of Michigan, Ann Arbor, MI, USA [2] Department of Pathology, CHA Bundang Medical Center, CHA University, Seongnam 463-712, Gyeonggi, Republic of Korea.

ABSTRACT
Somatic mutations or deletions of TP53 and PTEN in ductal carcinoma in situ lesions have been implicated in progression to invasive ductal carcinomas. A recent molecular and mutational analysis of breast cancers revealed that inactivation of tumor suppressors, p53 and PTEN, are strongly associated with triple negative breast cancer. In addition, these tumor suppressors have important roles in regulating self-renewal in normal and malignant stem cells. To investigate their role in breast carcinogenesis, we knocked down these genes in human mammary cells and in non-transformed MCF10A cells. p53 and PTEN knockdown synergized to activate pro-inflammatory interleukin-6 (IL6)/Stat3/nuclear factor κB signaling. This resulted in generation of highly metastatic epithelial-to-mesenchymal transition-like cancer stem cells resulting in tumors whose gene expression profile mimicked that found in basal/claudin-low molecular subtype within the triple negative breast tumors. Constitutive activation of this loop in transformed cells was dependent on proteolytic degradation of suppressor of cytokine signaling 3 (SOCS3) resulting in low levels of this protein in basal/claudin-low cell lines and primary tumors. In non-transformed cells, transient activation of the IL6 inflammatory loop induced SOCS3 expression leading to pathway inactivation. In transformed cells, enforced expression of SOCS3 or interfering with IL6 pathway via IL6R blockade inhibited tumor growth and metastasis in mouse xenograft models. Furthermore, circulating tumor cells were significantly reduced in tumor-bearing animals when treated with anti-IL6R antibodies. These studies uncover important connections between inflammation and carcinogenesis and suggest that blocking pro-inflammatory cytokines may be utilized as an attractive strategy to target triple negative breast tumors, which currently lacks molecularly targeted therapies.

Show MeSH
Related in: MedlinePlus