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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 cooperate to promote malignant properties in M6 cells, a HER-2-overexpressing mammary cancer model. (a) Anchorage independence was assessed by incubation of the indicated cell populations in soft agar for 14 days (N=3, bars, S.E.). (b) Anchorage independence of HMLE cells expressing ectopic KLF4/5 was determined as previously described. Empty vector (–) served as a control so that all cell populations were treated with equal volumes of lentiviral supernatant. (c) Cells were injected into the mammary gland of female athymic mice and tumor xenograft volume was monitored over a period of several weeks (left panel, N= 5; bars, S.E.). Similar effects on tumor burden were obtained using distinct shRNAs for the suppression of each KLF (right panel, N= 5; bars, S.E.). (d) Cell death was determined by Trypan blue exclusion following 24 h of matrix deprivation for the indicated cell populations (N=3; bars, S.E.). (e) As an independent method, cell death because of matrix deprivation in M6 cells was determined by fluorescence microscopic imaging of DAPI/PI-stained cells. Results were quantitated using ImageJ (two-tailed t-test; N=3; bars, S.E.). *P<0.05; **P<0.01; ***P<0.001
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fig4: KLF4/5 cooperate to promote malignant properties in M6 cells, a HER-2-overexpressing mammary cancer model. (a) Anchorage independence was assessed by incubation of the indicated cell populations in soft agar for 14 days (N=3, bars, S.E.). (b) Anchorage independence of HMLE cells expressing ectopic KLF4/5 was determined as previously described. Empty vector (–) served as a control so that all cell populations were treated with equal volumes of lentiviral supernatant. (c) Cells were injected into the mammary gland of female athymic mice and tumor xenograft volume was monitored over a period of several weeks (left panel, N= 5; bars, S.E.). Similar effects on tumor burden were obtained using distinct shRNAs for the suppression of each KLF (right panel, N= 5; bars, S.E.). (d) Cell death was determined by Trypan blue exclusion following 24 h of matrix deprivation for the indicated cell populations (N=3; bars, S.E.). (e) As an independent method, cell death because of matrix deprivation in M6 cells was determined by fluorescence microscopic imaging of DAPI/PI-stained cells. Results were quantitated using ImageJ (two-tailed t-test; N=3; bars, S.E.). *P<0.05; **P<0.01; ***P<0.001

Mentions: We next determined whether KLF4/5 contributed to malignant properties independently of lapatinib exposure. Relative to the control, depletion of either KLF4 or KLF5 in M6 cells significantly impacted anchorage-independent growth, as indicated by reduced colony-forming ability (Figure 4a). Furthermore, there was an additive reduction of colony number following codepletion. We similarly observed cooperativity in a gain-of-function context, as ectopic expression of KLF4/5 enhanced colony-forming ability in immortalized human breast epithelial cells (HMLE, Figure 4b).


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 cooperate to promote malignant properties in M6 cells, a HER-2-overexpressing mammary cancer model. (a) Anchorage independence was assessed by incubation of the indicated cell populations in soft agar for 14 days (N=3, bars, S.E.). (b) Anchorage independence of HMLE cells expressing ectopic KLF4/5 was determined as previously described. Empty vector (–) served as a control so that all cell populations were treated with equal volumes of lentiviral supernatant. (c) Cells were injected into the mammary gland of female athymic mice and tumor xenograft volume was monitored over a period of several weeks (left panel, N= 5; bars, S.E.). Similar effects on tumor burden were obtained using distinct shRNAs for the suppression of each KLF (right panel, N= 5; bars, S.E.). (d) Cell death was determined by Trypan blue exclusion following 24 h of matrix deprivation for the indicated cell populations (N=3; bars, S.E.). (e) As an independent method, cell death because of matrix deprivation in M6 cells was determined by fluorescence microscopic imaging of DAPI/PI-stained cells. Results were quantitated using ImageJ (two-tailed t-test; N=3; bars, S.E.). *P<0.05; **P<0.01; ***P<0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385942&req=5

fig4: KLF4/5 cooperate to promote malignant properties in M6 cells, a HER-2-overexpressing mammary cancer model. (a) Anchorage independence was assessed by incubation of the indicated cell populations in soft agar for 14 days (N=3, bars, S.E.). (b) Anchorage independence of HMLE cells expressing ectopic KLF4/5 was determined as previously described. Empty vector (–) served as a control so that all cell populations were treated with equal volumes of lentiviral supernatant. (c) Cells were injected into the mammary gland of female athymic mice and tumor xenograft volume was monitored over a period of several weeks (left panel, N= 5; bars, S.E.). Similar effects on tumor burden were obtained using distinct shRNAs for the suppression of each KLF (right panel, N= 5; bars, S.E.). (d) Cell death was determined by Trypan blue exclusion following 24 h of matrix deprivation for the indicated cell populations (N=3; bars, S.E.). (e) As an independent method, cell death because of matrix deprivation in M6 cells was determined by fluorescence microscopic imaging of DAPI/PI-stained cells. Results were quantitated using ImageJ (two-tailed t-test; N=3; bars, S.E.). *P<0.05; **P<0.01; ***P<0.001
Mentions: We next determined whether KLF4/5 contributed to malignant properties independently of lapatinib exposure. Relative to the control, depletion of either KLF4 or KLF5 in M6 cells significantly impacted anchorage-independent growth, as indicated by reduced colony-forming ability (Figure 4a). Furthermore, there was an additive reduction of colony number following codepletion. We similarly observed cooperativity in a gain-of-function context, as ectopic expression of KLF4/5 enhanced colony-forming ability in immortalized human breast epithelial cells (HMLE, Figure 4b).

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