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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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KLF4/5 depletion is associated with reduced expression of anti-apoptotic BCL2 family members. (a) BT474 cells were treated with DMSO, 1 μM lapatinib, sterile water or 10 μg/ml trastuzumab for the indicated time intervals. BCL2, BCL-XL and MCL1 levels were determined by western blot analysis. (b) Protein expression was analyzed in control or KLF-depleted BT474 and KPL4 cells. (c) Protein expression (left panel) and mRNA expression (right panel) was analyzed in the indicated cell populations. (d) Protein expression was analyzed in control HMLE cells and in cells expressing ectopic KLF4 and/or KLF5. (e) Spearman's correlation between KLF4, KLF5 and MCL1 levels as determined by RNAseq analysis of 890 human breast tumors. (f) The impact of KLF4/5 knockdown on the lapatinib-mediated induction of MCL1 was determined in BT474 cells. Cells were treated with 250 nM lapatinib or DMSO for 24 h. For three independent experiments, the expression levels were quantitated using ImageJ and normalized to β-actin (bars, S.D.). (g) MCL1 levels were reduced by siRNA and the resulting cell populations were treated with lapatinib for 96 h. For each cell population, cell viability relative to the DMSO control was obtained via ATP-based luminescent assay (paired t-test, two tailed; bars, S.D.). (h) Similarly, lapatinib resistance was analyzed in KLF4/5 knockdown BT474 cells following rescue with exogenous MCL1 expression vector or empty vector control. *P<0.05; **P<0.01; ***P<0.001
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fig5: KLF4/5 depletion is associated with reduced expression of anti-apoptotic BCL2 family members. (a) BT474 cells were treated with DMSO, 1 μM lapatinib, sterile water or 10 μg/ml trastuzumab for the indicated time intervals. BCL2, BCL-XL and MCL1 levels were determined by western blot analysis. (b) Protein expression was analyzed in control or KLF-depleted BT474 and KPL4 cells. (c) Protein expression (left panel) and mRNA expression (right panel) was analyzed in the indicated cell populations. (d) Protein expression was analyzed in control HMLE cells and in cells expressing ectopic KLF4 and/or KLF5. (e) Spearman's correlation between KLF4, KLF5 and MCL1 levels as determined by RNAseq analysis of 890 human breast tumors. (f) The impact of KLF4/5 knockdown on the lapatinib-mediated induction of MCL1 was determined in BT474 cells. Cells were treated with 250 nM lapatinib or DMSO for 24 h. For three independent experiments, the expression levels were quantitated using ImageJ and normalized to β-actin (bars, S.D.). (g) MCL1 levels were reduced by siRNA and the resulting cell populations were treated with lapatinib for 96 h. For each cell population, cell viability relative to the DMSO control was obtained via ATP-based luminescent assay (paired t-test, two tailed; bars, S.D.). (h) Similarly, lapatinib resistance was analyzed in KLF4/5 knockdown BT474 cells following rescue with exogenous MCL1 expression vector or empty vector control. *P<0.05; **P<0.01; ***P<0.001

Mentions: In response to lapatinib or trastuzumab treatment, we observed induction of not only KLF4/5 (Figures 3b and d), but also the anti-apoptotic BCL2 members BCL2, BCL-XL (BCL2L1) and MCL1 (Figure 5a). Although BCL2 was increased in response to lapatinib, BCL2 levels were decreased upon trastuzumab exposure, consistent with previous studies.45 Despite the well-documented oncogenic role for BCL2 in hematological malignancies, its expression is correlates with a favorable patient outcome in breast cancer.46


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)

KLF4/5 depletion is associated with reduced expression of anti-apoptotic BCL2 family members. (a) BT474 cells were treated with DMSO, 1 μM lapatinib, sterile water or 10 μg/ml trastuzumab for the indicated time intervals. BCL2, BCL-XL and MCL1 levels were determined by western blot analysis. (b) Protein expression was analyzed in control or KLF-depleted BT474 and KPL4 cells. (c) Protein expression (left panel) and mRNA expression (right panel) was analyzed in the indicated cell populations. (d) Protein expression was analyzed in control HMLE cells and in cells expressing ectopic KLF4 and/or KLF5. (e) Spearman's correlation between KLF4, KLF5 and MCL1 levels as determined by RNAseq analysis of 890 human breast tumors. (f) The impact of KLF4/5 knockdown on the lapatinib-mediated induction of MCL1 was determined in BT474 cells. Cells were treated with 250 nM lapatinib or DMSO for 24 h. For three independent experiments, the expression levels were quantitated using ImageJ and normalized to β-actin (bars, S.D.). (g) MCL1 levels were reduced by siRNA and the resulting cell populations were treated with lapatinib for 96 h. For each cell population, cell viability relative to the DMSO control was obtained via ATP-based luminescent assay (paired t-test, two tailed; bars, S.D.). (h) Similarly, lapatinib resistance was analyzed in KLF4/5 knockdown BT474 cells following rescue with exogenous MCL1 expression vector or empty vector control. *P<0.05; **P<0.01; ***P<0.001
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fig5: KLF4/5 depletion is associated with reduced expression of anti-apoptotic BCL2 family members. (a) BT474 cells were treated with DMSO, 1 μM lapatinib, sterile water or 10 μg/ml trastuzumab for the indicated time intervals. BCL2, BCL-XL and MCL1 levels were determined by western blot analysis. (b) Protein expression was analyzed in control or KLF-depleted BT474 and KPL4 cells. (c) Protein expression (left panel) and mRNA expression (right panel) was analyzed in the indicated cell populations. (d) Protein expression was analyzed in control HMLE cells and in cells expressing ectopic KLF4 and/or KLF5. (e) Spearman's correlation between KLF4, KLF5 and MCL1 levels as determined by RNAseq analysis of 890 human breast tumors. (f) The impact of KLF4/5 knockdown on the lapatinib-mediated induction of MCL1 was determined in BT474 cells. Cells were treated with 250 nM lapatinib or DMSO for 24 h. For three independent experiments, the expression levels were quantitated using ImageJ and normalized to β-actin (bars, S.D.). (g) MCL1 levels were reduced by siRNA and the resulting cell populations were treated with lapatinib for 96 h. For each cell population, cell viability relative to the DMSO control was obtained via ATP-based luminescent assay (paired t-test, two tailed; bars, S.D.). (h) Similarly, lapatinib resistance was analyzed in KLF4/5 knockdown BT474 cells following rescue with exogenous MCL1 expression vector or empty vector control. *P<0.05; **P<0.01; ***P<0.001
Mentions: In response to lapatinib or trastuzumab treatment, we observed induction of not only KLF4/5 (Figures 3b and d), but also the anti-apoptotic BCL2 members BCL2, BCL-XL (BCL2L1) and MCL1 (Figure 5a). Although BCL2 was increased in response to lapatinib, BCL2 levels were decreased upon trastuzumab exposure, consistent with previous studies.45 Despite the well-documented oncogenic role for BCL2 in hematological malignancies, its expression is correlates with a favorable patient outcome in breast cancer.46

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