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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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Endogenous KLF4/5 mediate lapatinib resistance in breast cancer models. (a) Levels of KLF4/5 in primary human breast tumors were determined by RNAseq (Illumina HiSeq RNAseqV2). Upper quartile normalized data were downloaded from TCGA and assigned a PAM50 subtype. Spearman's correlation was performed on the log2 transformed data. (b) BT474 cells were treated with DMSO or lapatinib for the indicated interval. Whole-cell lysate was analyzed by western blot. Expression levels from three independent experiments were determined using ImageJ for quantitation, with normalization to β-actin (bars, S.D.). (c) BT474 cells were treated with trastuzumab or sterile water for the indicated interval and whole-cell lysate was analyzed by western blot. (d) KLF4/5 transcript levels were determined by qRT-PCR following lapatinib exposure. Expression data were normalized using the housekeeping gene B2M. (e) The pMIR-Report-Luc-KLF4-FL translation reporter contains as an insert within the FLuc 3' UTR the full-length KLF4 transcript, including the KLF4 protein coding region and the flanking UTRs, as previously described.18 Translation efficiency was measured by determining normalized Fluc activity in BT474 cells treated for 24 h with lapatinib or DMSO (left panel). miR-206 levels were determined by qRT-PCR following 24-h lapatinib exposure. Expression data were normalized using U6 snRNA (right panel). (f) Cells were treated with the indicated shRNA construct, 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 luminescence assay (bars, S.D.). (g) Similarly, the lapatinib effect on the relative cell viability of M6 cells expressing ectopic KLF4 and/or KLF5 was determined. Empty vector served as a control. (h) To assess MMI, M6 cells were treated with lapatinib for 24 h, stained with 250 nM of Mitotracker dye and analyzed by flow cytometry. (i) To assess activity of the intrinsic apoptotic pathway, caspase-9 levels were determined in M6 cells expressing shCtl, shKLF4, shKLF5 or shKLF4/5. Cells were treated with lapatinib for 24 h before preparation of cell extracts. *P<0.05; **P<0.01; ***P<0.001
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fig3: Endogenous KLF4/5 mediate lapatinib resistance in breast cancer models. (a) Levels of KLF4/5 in primary human breast tumors were determined by RNAseq (Illumina HiSeq RNAseqV2). Upper quartile normalized data were downloaded from TCGA and assigned a PAM50 subtype. Spearman's correlation was performed on the log2 transformed data. (b) BT474 cells were treated with DMSO or lapatinib for the indicated interval. Whole-cell lysate was analyzed by western blot. Expression levels from three independent experiments were determined using ImageJ for quantitation, with normalization to β-actin (bars, S.D.). (c) BT474 cells were treated with trastuzumab or sterile water for the indicated interval and whole-cell lysate was analyzed by western blot. (d) KLF4/5 transcript levels were determined by qRT-PCR following lapatinib exposure. Expression data were normalized using the housekeeping gene B2M. (e) The pMIR-Report-Luc-KLF4-FL translation reporter contains as an insert within the FLuc 3' UTR the full-length KLF4 transcript, including the KLF4 protein coding region and the flanking UTRs, as previously described.18 Translation efficiency was measured by determining normalized Fluc activity in BT474 cells treated for 24 h with lapatinib or DMSO (left panel). miR-206 levels were determined by qRT-PCR following 24-h lapatinib exposure. Expression data were normalized using U6 snRNA (right panel). (f) Cells were treated with the indicated shRNA construct, 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 luminescence assay (bars, S.D.). (g) Similarly, the lapatinib effect on the relative cell viability of M6 cells expressing ectopic KLF4 and/or KLF5 was determined. Empty vector served as a control. (h) To assess MMI, M6 cells were treated with lapatinib for 24 h, stained with 250 nM of Mitotracker dye and analyzed by flow cytometry. (i) To assess activity of the intrinsic apoptotic pathway, caspase-9 levels were determined in M6 cells expressing shCtl, shKLF4, shKLF5 or shKLF4/5. Cells were treated with lapatinib for 24 h before preparation of cell extracts. *P<0.05; **P<0.01; ***P<0.001

Mentions: We next examined the transcript abundance of KLF4/5 as determined by RNAseq analysis of patient breast tumors via The Cancer Genome Atlas (TCGA) Research Network. Among the breast cancer intrinsic subtypes, we observed the expression of the two factors to be most highly correlated in HER2-enriched tumors (Figure 3a and Supplementary Figure 2). Given that KLF4/5 appeared to represent positively correlated prognostic factors in the HER2-enriched breast cancer subtype, we subsequently investigated the interdependence of KLF4/5 expression with exposure to, or resistance to, HER2-targeted therapy.


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)

Endogenous KLF4/5 mediate lapatinib resistance in breast cancer models. (a) Levels of KLF4/5 in primary human breast tumors were determined by RNAseq (Illumina HiSeq RNAseqV2). Upper quartile normalized data were downloaded from TCGA and assigned a PAM50 subtype. Spearman's correlation was performed on the log2 transformed data. (b) BT474 cells were treated with DMSO or lapatinib for the indicated interval. Whole-cell lysate was analyzed by western blot. Expression levels from three independent experiments were determined using ImageJ for quantitation, with normalization to β-actin (bars, S.D.). (c) BT474 cells were treated with trastuzumab or sterile water for the indicated interval and whole-cell lysate was analyzed by western blot. (d) KLF4/5 transcript levels were determined by qRT-PCR following lapatinib exposure. Expression data were normalized using the housekeeping gene B2M. (e) The pMIR-Report-Luc-KLF4-FL translation reporter contains as an insert within the FLuc 3' UTR the full-length KLF4 transcript, including the KLF4 protein coding region and the flanking UTRs, as previously described.18 Translation efficiency was measured by determining normalized Fluc activity in BT474 cells treated for 24 h with lapatinib or DMSO (left panel). miR-206 levels were determined by qRT-PCR following 24-h lapatinib exposure. Expression data were normalized using U6 snRNA (right panel). (f) Cells were treated with the indicated shRNA construct, 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 luminescence assay (bars, S.D.). (g) Similarly, the lapatinib effect on the relative cell viability of M6 cells expressing ectopic KLF4 and/or KLF5 was determined. Empty vector served as a control. (h) To assess MMI, M6 cells were treated with lapatinib for 24 h, stained with 250 nM of Mitotracker dye and analyzed by flow cytometry. (i) To assess activity of the intrinsic apoptotic pathway, caspase-9 levels were determined in M6 cells expressing shCtl, shKLF4, shKLF5 or shKLF4/5. Cells were treated with lapatinib for 24 h before preparation of cell extracts. *P<0.05; **P<0.01; ***P<0.001
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Related In: Results  -  Collection

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fig3: Endogenous KLF4/5 mediate lapatinib resistance in breast cancer models. (a) Levels of KLF4/5 in primary human breast tumors were determined by RNAseq (Illumina HiSeq RNAseqV2). Upper quartile normalized data were downloaded from TCGA and assigned a PAM50 subtype. Spearman's correlation was performed on the log2 transformed data. (b) BT474 cells were treated with DMSO or lapatinib for the indicated interval. Whole-cell lysate was analyzed by western blot. Expression levels from three independent experiments were determined using ImageJ for quantitation, with normalization to β-actin (bars, S.D.). (c) BT474 cells were treated with trastuzumab or sterile water for the indicated interval and whole-cell lysate was analyzed by western blot. (d) KLF4/5 transcript levels were determined by qRT-PCR following lapatinib exposure. Expression data were normalized using the housekeeping gene B2M. (e) The pMIR-Report-Luc-KLF4-FL translation reporter contains as an insert within the FLuc 3' UTR the full-length KLF4 transcript, including the KLF4 protein coding region and the flanking UTRs, as previously described.18 Translation efficiency was measured by determining normalized Fluc activity in BT474 cells treated for 24 h with lapatinib or DMSO (left panel). miR-206 levels were determined by qRT-PCR following 24-h lapatinib exposure. Expression data were normalized using U6 snRNA (right panel). (f) Cells were treated with the indicated shRNA construct, 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 luminescence assay (bars, S.D.). (g) Similarly, the lapatinib effect on the relative cell viability of M6 cells expressing ectopic KLF4 and/or KLF5 was determined. Empty vector served as a control. (h) To assess MMI, M6 cells were treated with lapatinib for 24 h, stained with 250 nM of Mitotracker dye and analyzed by flow cytometry. (i) To assess activity of the intrinsic apoptotic pathway, caspase-9 levels were determined in M6 cells expressing shCtl, shKLF4, shKLF5 or shKLF4/5. Cells were treated with lapatinib for 24 h before preparation of cell extracts. *P<0.05; **P<0.01; ***P<0.001
Mentions: We next examined the transcript abundance of KLF4/5 as determined by RNAseq analysis of patient breast tumors via The Cancer Genome Atlas (TCGA) Research Network. Among the breast cancer intrinsic subtypes, we observed the expression of the two factors to be most highly correlated in HER2-enriched tumors (Figure 3a and Supplementary Figure 2). Given that KLF4/5 appeared to represent positively correlated prognostic factors in the HER2-enriched breast cancer subtype, we subsequently investigated the interdependence of KLF4/5 expression with exposure to, or resistance to, HER2-targeted therapy.

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