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c-Jun N-terminal kinase 2 prevents luminal cell commitment in normal mammary glands and tumors by inhibiting p53/Notch1 and breast cancer gene 1 expression.

Cantrell MA, Ebelt ND, Pfefferle AD, Perou CM, Van Den Berg CL - Oncotarget (2015)

Bottom Line: Mechanisms governing mammary tumor subtype generation could prove critical to developing better treatments.In a p53 knockout model, JNK2 restricts luminal populations independently of Notch1, by suppressing Brca1 expression and promoting epithelial to mesenchymal transition.JNK2 also inhibits estrogen receptor (ER) expression and confers resistance to fulvestrant, an ER inhibitor, while stimulating tumor progression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cellular & Molecular Biology, College of Pharmacy, University of Texas at Austin, Dell Pediatric Research Institute, Austin, TX 78723, USA.

ABSTRACT
Breast cancer is a heterogeneous disease with several subtypes carrying unique prognoses. Patients with differentiated luminal tumors experience better outcomes, while effective treatments are unavailable for poorly differentiated tumors, including the basal-like subtype. Mechanisms governing mammary tumor subtype generation could prove critical to developing better treatments. C-Jun N-terminal kinase 2 (JNK2) is important in mammary tumorigenesis and tumor progression. Using a variety of mouse models, human breast cancer cell lines and tumor expression data, studies herein support that JNK2 inhibits cell differentiation in normal and cancer-derived mammary cells. JNK2 prevents precocious pubertal mammary development and inhibits Notch-dependent expansion of luminal cell populations. Likewise, JNK2 suppresses luminal populations in a p53-competent Polyoma Middle T-antigen tumor model where jnk2 knockout causes p53-dependent upregulation of Notch1 transcription. In a p53 knockout model, JNK2 restricts luminal populations independently of Notch1, by suppressing Brca1 expression and promoting epithelial to mesenchymal transition. JNK2 also inhibits estrogen receptor (ER) expression and confers resistance to fulvestrant, an ER inhibitor, while stimulating tumor progression. These data suggest that therapies inhibiting JNK2 in breast cancer may promote tumor differentiation, improve endocrine therapy response, and inhibit metastasis.

No MeSH data available.


Related in: MedlinePlus

Absence of jnk2 in MT tumors increases luminal cell populations and enhances p53 and Notch1 expressionA-B. Representative images and quantification of p63+ A or CK8/18+ B cells in MT tumors (n = 5 per genotype); C. Representative images of Notch1ICD+ cells in MT tumors; D. Notch1 expression by qPCR in MT tumors; E-F.Hes1 E and Notch1F expression by qPCR in MT/jnk2ko cells; G. Western blot of full-length Notch1 in MT/jnk2ko GFP and GFP-JNK2 cells; H. p53 expression in MT/jnk2ko GFP and GFP-JNK2 cells by qPCR; I.p53 expression in normal mammary organoids by qPCR; J. MT/jnk2ko cells were transfected with Notch1 promoter constructs and analyzed for promoter activity (N1PR= wildtype Notch1 promoter, N1PRp53mut = Notch1 promoter with mutated p53 response elements, N1PRless = promoter-less control plasmid); K. Chromatin Immunoprecipitation assay of p53 binding to p53 response elements within the p21 and Notch1 promoters in MT/jnk2ko cells. Gapdh promoter used as negative control. 1 Way ANOVA with post-hoc t-test was performed for J followed by post-hoc t-test. Nonparametric t-test was performed for all others, *p < 0.05, **p < 0.001.
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Figure 3: Absence of jnk2 in MT tumors increases luminal cell populations and enhances p53 and Notch1 expressionA-B. Representative images and quantification of p63+ A or CK8/18+ B cells in MT tumors (n = 5 per genotype); C. Representative images of Notch1ICD+ cells in MT tumors; D. Notch1 expression by qPCR in MT tumors; E-F.Hes1 E and Notch1F expression by qPCR in MT/jnk2ko cells; G. Western blot of full-length Notch1 in MT/jnk2ko GFP and GFP-JNK2 cells; H. p53 expression in MT/jnk2ko GFP and GFP-JNK2 cells by qPCR; I.p53 expression in normal mammary organoids by qPCR; J. MT/jnk2ko cells were transfected with Notch1 promoter constructs and analyzed for promoter activity (N1PR= wildtype Notch1 promoter, N1PRp53mut = Notch1 promoter with mutated p53 response elements, N1PRless = promoter-less control plasmid); K. Chromatin Immunoprecipitation assay of p53 binding to p53 response elements within the p21 and Notch1 promoters in MT/jnk2ko cells. Gapdh promoter used as negative control. 1 Way ANOVA with post-hoc t-test was performed for J followed by post-hoc t-test. Nonparametric t-test was performed for all others, *p < 0.05, **p < 0.001.

Mentions: The importance of decisive differentiation pathways in the normal gland in tumor differentiation is illustrated by the finding that basal-type tumors may originate from luminal progenitors rather than basal/myoepithelial cells [2]. To explore whether JNK2 affects tumor cell differentiation, we used an MT transgenic mouse model. MT tumors most closely reflect the phenotypic characteristics of human luminal breast cancer [21], [22]. MT/jnk2wt and MT/jnk2ko mammary tumors were immunostained with p63 or CK8/18 antibodies to identify basal and luminal cells, respectively. Although a high proportion of tumor cells do not express either marker, MT/jnk2ko tumors have significantly fewer p63+ nuclei and more CK8/18+ cells (Fig 3A and 3B, p = 0.0079 and p = 0.0411 respectively). Moreover, MT/jnk2ko tumors express Notch1ICD in almost all cells, whereas MT/jnk2wt tumors are nearly void of its expression, (Fig 3C). Correspondingly, MT/jnk2ko tumors express four-fold higher Notch1 mRNA (Fig 3D, p = 0.0101). These results suggest that, akin to the normal mammary gland, JNK2 inhibits luminal cell populations in the spontaneous MT tumor model.


c-Jun N-terminal kinase 2 prevents luminal cell commitment in normal mammary glands and tumors by inhibiting p53/Notch1 and breast cancer gene 1 expression.

Cantrell MA, Ebelt ND, Pfefferle AD, Perou CM, Van Den Berg CL - Oncotarget (2015)

Absence of jnk2 in MT tumors increases luminal cell populations and enhances p53 and Notch1 expressionA-B. Representative images and quantification of p63+ A or CK8/18+ B cells in MT tumors (n = 5 per genotype); C. Representative images of Notch1ICD+ cells in MT tumors; D. Notch1 expression by qPCR in MT tumors; E-F.Hes1 E and Notch1F expression by qPCR in MT/jnk2ko cells; G. Western blot of full-length Notch1 in MT/jnk2ko GFP and GFP-JNK2 cells; H. p53 expression in MT/jnk2ko GFP and GFP-JNK2 cells by qPCR; I.p53 expression in normal mammary organoids by qPCR; J. MT/jnk2ko cells were transfected with Notch1 promoter constructs and analyzed for promoter activity (N1PR= wildtype Notch1 promoter, N1PRp53mut = Notch1 promoter with mutated p53 response elements, N1PRless = promoter-less control plasmid); K. Chromatin Immunoprecipitation assay of p53 binding to p53 response elements within the p21 and Notch1 promoters in MT/jnk2ko cells. Gapdh promoter used as negative control. 1 Way ANOVA with post-hoc t-test was performed for J followed by post-hoc t-test. Nonparametric t-test was performed for all others, *p < 0.05, **p < 0.001.
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Figure 3: Absence of jnk2 in MT tumors increases luminal cell populations and enhances p53 and Notch1 expressionA-B. Representative images and quantification of p63+ A or CK8/18+ B cells in MT tumors (n = 5 per genotype); C. Representative images of Notch1ICD+ cells in MT tumors; D. Notch1 expression by qPCR in MT tumors; E-F.Hes1 E and Notch1F expression by qPCR in MT/jnk2ko cells; G. Western blot of full-length Notch1 in MT/jnk2ko GFP and GFP-JNK2 cells; H. p53 expression in MT/jnk2ko GFP and GFP-JNK2 cells by qPCR; I.p53 expression in normal mammary organoids by qPCR; J. MT/jnk2ko cells were transfected with Notch1 promoter constructs and analyzed for promoter activity (N1PR= wildtype Notch1 promoter, N1PRp53mut = Notch1 promoter with mutated p53 response elements, N1PRless = promoter-less control plasmid); K. Chromatin Immunoprecipitation assay of p53 binding to p53 response elements within the p21 and Notch1 promoters in MT/jnk2ko cells. Gapdh promoter used as negative control. 1 Way ANOVA with post-hoc t-test was performed for J followed by post-hoc t-test. Nonparametric t-test was performed for all others, *p < 0.05, **p < 0.001.
Mentions: The importance of decisive differentiation pathways in the normal gland in tumor differentiation is illustrated by the finding that basal-type tumors may originate from luminal progenitors rather than basal/myoepithelial cells [2]. To explore whether JNK2 affects tumor cell differentiation, we used an MT transgenic mouse model. MT tumors most closely reflect the phenotypic characteristics of human luminal breast cancer [21], [22]. MT/jnk2wt and MT/jnk2ko mammary tumors were immunostained with p63 or CK8/18 antibodies to identify basal and luminal cells, respectively. Although a high proportion of tumor cells do not express either marker, MT/jnk2ko tumors have significantly fewer p63+ nuclei and more CK8/18+ cells (Fig 3A and 3B, p = 0.0079 and p = 0.0411 respectively). Moreover, MT/jnk2ko tumors express Notch1ICD in almost all cells, whereas MT/jnk2wt tumors are nearly void of its expression, (Fig 3C). Correspondingly, MT/jnk2ko tumors express four-fold higher Notch1 mRNA (Fig 3D, p = 0.0101). These results suggest that, akin to the normal mammary gland, JNK2 inhibits luminal cell populations in the spontaneous MT tumor model.

Bottom Line: Mechanisms governing mammary tumor subtype generation could prove critical to developing better treatments.In a p53 knockout model, JNK2 restricts luminal populations independently of Notch1, by suppressing Brca1 expression and promoting epithelial to mesenchymal transition.JNK2 also inhibits estrogen receptor (ER) expression and confers resistance to fulvestrant, an ER inhibitor, while stimulating tumor progression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cellular & Molecular Biology, College of Pharmacy, University of Texas at Austin, Dell Pediatric Research Institute, Austin, TX 78723, USA.

ABSTRACT
Breast cancer is a heterogeneous disease with several subtypes carrying unique prognoses. Patients with differentiated luminal tumors experience better outcomes, while effective treatments are unavailable for poorly differentiated tumors, including the basal-like subtype. Mechanisms governing mammary tumor subtype generation could prove critical to developing better treatments. C-Jun N-terminal kinase 2 (JNK2) is important in mammary tumorigenesis and tumor progression. Using a variety of mouse models, human breast cancer cell lines and tumor expression data, studies herein support that JNK2 inhibits cell differentiation in normal and cancer-derived mammary cells. JNK2 prevents precocious pubertal mammary development and inhibits Notch-dependent expansion of luminal cell populations. Likewise, JNK2 suppresses luminal populations in a p53-competent Polyoma Middle T-antigen tumor model where jnk2 knockout causes p53-dependent upregulation of Notch1 transcription. In a p53 knockout model, JNK2 restricts luminal populations independently of Notch1, by suppressing Brca1 expression and promoting epithelial to mesenchymal transition. JNK2 also inhibits estrogen receptor (ER) expression and confers resistance to fulvestrant, an ER inhibitor, while stimulating tumor progression. These data suggest that therapies inhibiting JNK2 in breast cancer may promote tumor differentiation, improve endocrine therapy response, and inhibit metastasis.

No MeSH data available.


Related in: MedlinePlus