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Regulation of anti-apoptotic signaling by Kruppel-like factors 4 and 5 mediates lapatinib resistance in breast cancer.

Farrugia MK, Sharma SB, Lin CC, McLaughlin SL, Vanderbilt DB, Ammer AG, Salkeni MA, Stoilov P, Agazie YM, Creighton CJ, Ruppert JM - Cell Death Dis (2015)

Bottom Line: Indicating cooperativity, greater effects were observed when both genes were depleted.KLF4/5-deficient cells had reduced basal mRNA and protein levels of the anti-apoptotic factors myeloid cell leukemia 1 (MCL1) and B-cell lymphoma-extra large (BCL-XL).These results identify KLF4 and KLF5 as cooperating protumorigenic factors and critical participants in resistance to lapatinib, furthering the rationale for combining anti-MCL1/BCL-XL inhibitors with conventional HER2-targeted therapies.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, West Virginia University Health Sciences Center, Morgantown, WV 26506, USA [2] Program in Cancer Cell Biology, West Virginia University, Morgantown, WV 26506, USA.

ABSTRACT
The Kruppel-like transcription factors (KLFs) 4 and 5 (KLF4/5) are coexpressed in mouse embryonic stem cells, where they function redundantly to maintain pluripotency. In mammary carcinoma, KLF4/5 can each impact the malignant phenotype, but potential linkages to drug resistance remain unclear. In primary human breast cancers, we observed a positive correlation between KLF4/5 transcript abundance, particularly in the human epidermal growth factor receptor 2 (HER2)-enriched subtype. Furthermore, KLF4/5 protein was rapidly upregulated in human breast cancer cells following treatment with the HER2/epidermal growth factor receptor inhibitor, lapatinib. In addition, we observed a positive correlation between these factors in the primary tumors of genetically engineered mouse models (GEMMs). In particular, the levels of both factors were enriched in the basal-like tumors of the C3(1) TAg (SV40 large T antigen transgenic mice under control of the C3(1)/prostatein promoter) GEMM. Using tumor cells derived from this model as well as human breast cancer cells, suppression of KLF4 and/or KLF5 sensitized HER2-overexpressing cells to lapatinib. Indicating cooperativity, greater effects were observed when both genes were depleted. KLF4/5-deficient cells had reduced basal mRNA and protein levels of the anti-apoptotic factors myeloid cell leukemia 1 (MCL1) and B-cell lymphoma-extra large (BCL-XL). Moreover, MCL1 was upregulated by lapatinib in a KLF4/5-dependent manner, and enforced expression of MCL1 in KLF4/5-deficient cells restored drug resistance. In addition, combined suppression of KLF4/5 in cultured tumor cells additively inhibited anchorage-independent growth, resistance to anoikis and tumor formation in immunocompromised mice. Consistent with their cooperative role in drug resistance and other malignant properties, KLF4/5 levels selectively stratified human HER2-enriched breast cancer by distant metastasis-free survival. These results identify KLF4 and KLF5 as cooperating protumorigenic factors and critical participants in resistance to lapatinib, furthering the rationale for combining anti-MCL1/BCL-XL inhibitors with conventional HER2-targeted therapies.

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Klf4 and Klf5 are differentially expressed and positively correlated in GEMMs of breast cancer. (a) Microarray analysis of Klf4/5 levels across GEMMs of breast cancer. Data for 58 mammary tumors from the Gene Expression Omnibus (GSE3165) were organized by GEMM and molecular subtype. Expression values were normalized to whole mouse RNA (bars, S.D.). Klf4 levels in luminal and non-luminal tumors were compared via one-way ANOVA using Dunnet's post test (P<0.0001). (b) Spearman's correlation was performed for the samples in panel a. (c) qRT-PCR analysis of total RNA isolated from tumors of MMTV-Neu or C3(1) TAg transgenic mice (left panel). Normal mammary tissue from FVB/N mice was analyzed similarly (NL breast, N=3). Tumor mean expression is depicted relative to the mean for normal tissue (bars, S.E.). The overall mean tumor expression of Klf4 and Klf5 was compared between GEMMs using a two-tailed t-test (for each gene, P<0.0001). The log2 transformed data were assessed by Spearman's correlation (right panel). (d) Western blot analysis of KLF4/5 levels in whole-cell lysate of 10 different breast cancer cell lines. KLF expression was determined using ImageJ and normalized to β-actin. The expression values were assessed by Spearman's correlation (right panel)
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fig1: Klf4 and Klf5 are differentially expressed and positively correlated in GEMMs of breast cancer. (a) Microarray analysis of Klf4/5 levels across GEMMs of breast cancer. Data for 58 mammary tumors from the Gene Expression Omnibus (GSE3165) were organized by GEMM and molecular subtype. Expression values were normalized to whole mouse RNA (bars, S.D.). Klf4 levels in luminal and non-luminal tumors were compared via one-way ANOVA using Dunnet's post test (P<0.0001). (b) Spearman's correlation was performed for the samples in panel a. (c) qRT-PCR analysis of total RNA isolated from tumors of MMTV-Neu or C3(1) TAg transgenic mice (left panel). Normal mammary tissue from FVB/N mice was analyzed similarly (NL breast, N=3). Tumor mean expression is depicted relative to the mean for normal tissue (bars, S.E.). The overall mean tumor expression of Klf4 and Klf5 was compared between GEMMs using a two-tailed t-test (for each gene, P<0.0001). The log2 transformed data were assessed by Spearman's correlation (right panel). (d) Western blot analysis of KLF4/5 levels in whole-cell lysate of 10 different breast cancer cell lines. KLF expression was determined using ImageJ and normalized to β-actin. The expression values were assessed by Spearman's correlation (right panel)

Mentions: Of 108 tumors in the microarray data set, we analyzed 58 tumor samples across 9 different GEMMs.38 We omitted models that had very low abundance of Klf4/5, including p53-deficient models and models on the BALB/c background. We also omitted samples that did not cluster into an intrinsic subtype (14 tumor samples), and we excluded the mesenchymal subgroup tumors because of heterogeneity in GEMM of origin (five tumors) (Figure 1a). Unlike for Klf5, Klf4 expression as determined by microarray analysis varied substantially across the mouse model tumors, with higher expression in mice transgenic for the coding region of SV40 large T antigen driven by the C3(1)/prostatein promoter (i.e., C3(1) TAg) than in mouse mammary tumor virus promoter (MMTV)-Neu tumors. Collectively, Klf4 levels were significantly lower in GEMMs that generated predominantly luminal tumors relative to tumors with basal characteristics (P<0.0001).


Regulation of anti-apoptotic signaling by Kruppel-like factors 4 and 5 mediates lapatinib resistance in breast cancer.

Farrugia MK, Sharma SB, Lin CC, McLaughlin SL, Vanderbilt DB, Ammer AG, Salkeni MA, Stoilov P, Agazie YM, Creighton CJ, Ruppert JM - Cell Death Dis (2015)

Klf4 and Klf5 are differentially expressed and positively correlated in GEMMs of breast cancer. (a) Microarray analysis of Klf4/5 levels across GEMMs of breast cancer. Data for 58 mammary tumors from the Gene Expression Omnibus (GSE3165) were organized by GEMM and molecular subtype. Expression values were normalized to whole mouse RNA (bars, S.D.). Klf4 levels in luminal and non-luminal tumors were compared via one-way ANOVA using Dunnet's post test (P<0.0001). (b) Spearman's correlation was performed for the samples in panel a. (c) qRT-PCR analysis of total RNA isolated from tumors of MMTV-Neu or C3(1) TAg transgenic mice (left panel). Normal mammary tissue from FVB/N mice was analyzed similarly (NL breast, N=3). Tumor mean expression is depicted relative to the mean for normal tissue (bars, S.E.). The overall mean tumor expression of Klf4 and Klf5 was compared between GEMMs using a two-tailed t-test (for each gene, P<0.0001). The log2 transformed data were assessed by Spearman's correlation (right panel). (d) Western blot analysis of KLF4/5 levels in whole-cell lysate of 10 different breast cancer cell lines. KLF expression was determined using ImageJ and normalized to β-actin. The expression values were assessed by Spearman's correlation (right panel)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4385942&req=5

fig1: Klf4 and Klf5 are differentially expressed and positively correlated in GEMMs of breast cancer. (a) Microarray analysis of Klf4/5 levels across GEMMs of breast cancer. Data for 58 mammary tumors from the Gene Expression Omnibus (GSE3165) were organized by GEMM and molecular subtype. Expression values were normalized to whole mouse RNA (bars, S.D.). Klf4 levels in luminal and non-luminal tumors were compared via one-way ANOVA using Dunnet's post test (P<0.0001). (b) Spearman's correlation was performed for the samples in panel a. (c) qRT-PCR analysis of total RNA isolated from tumors of MMTV-Neu or C3(1) TAg transgenic mice (left panel). Normal mammary tissue from FVB/N mice was analyzed similarly (NL breast, N=3). Tumor mean expression is depicted relative to the mean for normal tissue (bars, S.E.). The overall mean tumor expression of Klf4 and Klf5 was compared between GEMMs using a two-tailed t-test (for each gene, P<0.0001). The log2 transformed data were assessed by Spearman's correlation (right panel). (d) Western blot analysis of KLF4/5 levels in whole-cell lysate of 10 different breast cancer cell lines. KLF expression was determined using ImageJ and normalized to β-actin. The expression values were assessed by Spearman's correlation (right panel)
Mentions: Of 108 tumors in the microarray data set, we analyzed 58 tumor samples across 9 different GEMMs.38 We omitted models that had very low abundance of Klf4/5, including p53-deficient models and models on the BALB/c background. We also omitted samples that did not cluster into an intrinsic subtype (14 tumor samples), and we excluded the mesenchymal subgroup tumors because of heterogeneity in GEMM of origin (five tumors) (Figure 1a). Unlike for Klf5, Klf4 expression as determined by microarray analysis varied substantially across the mouse model tumors, with higher expression in mice transgenic for the coding region of SV40 large T antigen driven by the C3(1)/prostatein promoter (i.e., C3(1) TAg) than in mouse mammary tumor virus promoter (MMTV)-Neu tumors. Collectively, Klf4 levels were significantly lower in GEMMs that generated predominantly luminal tumors relative to tumors with basal characteristics (P<0.0001).

Bottom Line: Indicating cooperativity, greater effects were observed when both genes were depleted.KLF4/5-deficient cells had reduced basal mRNA and protein levels of the anti-apoptotic factors myeloid cell leukemia 1 (MCL1) and B-cell lymphoma-extra large (BCL-XL).These results identify KLF4 and KLF5 as cooperating protumorigenic factors and critical participants in resistance to lapatinib, furthering the rationale for combining anti-MCL1/BCL-XL inhibitors with conventional HER2-targeted therapies.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, West Virginia University Health Sciences Center, Morgantown, WV 26506, USA [2] Program in Cancer Cell Biology, West Virginia University, Morgantown, WV 26506, USA.

ABSTRACT
The Kruppel-like transcription factors (KLFs) 4 and 5 (KLF4/5) are coexpressed in mouse embryonic stem cells, where they function redundantly to maintain pluripotency. In mammary carcinoma, KLF4/5 can each impact the malignant phenotype, but potential linkages to drug resistance remain unclear. In primary human breast cancers, we observed a positive correlation between KLF4/5 transcript abundance, particularly in the human epidermal growth factor receptor 2 (HER2)-enriched subtype. Furthermore, KLF4/5 protein was rapidly upregulated in human breast cancer cells following treatment with the HER2/epidermal growth factor receptor inhibitor, lapatinib. In addition, we observed a positive correlation between these factors in the primary tumors of genetically engineered mouse models (GEMMs). In particular, the levels of both factors were enriched in the basal-like tumors of the C3(1) TAg (SV40 large T antigen transgenic mice under control of the C3(1)/prostatein promoter) GEMM. Using tumor cells derived from this model as well as human breast cancer cells, suppression of KLF4 and/or KLF5 sensitized HER2-overexpressing cells to lapatinib. Indicating cooperativity, greater effects were observed when both genes were depleted. KLF4/5-deficient cells had reduced basal mRNA and protein levels of the anti-apoptotic factors myeloid cell leukemia 1 (MCL1) and B-cell lymphoma-extra large (BCL-XL). Moreover, MCL1 was upregulated by lapatinib in a KLF4/5-dependent manner, and enforced expression of MCL1 in KLF4/5-deficient cells restored drug resistance. In addition, combined suppression of KLF4/5 in cultured tumor cells additively inhibited anchorage-independent growth, resistance to anoikis and tumor formation in immunocompromised mice. Consistent with their cooperative role in drug resistance and other malignant properties, KLF4/5 levels selectively stratified human HER2-enriched breast cancer by distant metastasis-free survival. These results identify KLF4 and KLF5 as cooperating protumorigenic factors and critical participants in resistance to lapatinib, furthering the rationale for combining anti-MCL1/BCL-XL inhibitors with conventional HER2-targeted therapies.

Show MeSH
Related in: MedlinePlus