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. 2015 Mar 19;6(3):e1699.
doi: 10.1038/cddis.2015.65.

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

M K Farrugia  1 , S B Sharma  1 , C-C Lin  2 , S L McLaughlin  3 , D B Vanderbilt  1 , A G Ammer  3 , M A Salkeni  4 , P Stoilov  5 , Y M Agazie  5 , C J Creighton  6 , J M Ruppert  5
Affiliations

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

M K Farrugia et al. Cell Death Dis. .

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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Figures

Figure 1
Figure 1
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)
Figure 2
Figure 2
Prognostic significance of KLF4 and KLF5 in human breast cancer. Kaplan–Meier analysis utilized a previously described breast cancer microarray database. Red (hi) and blue (lo) groups were defined using the median gene expression level within the tumors of breast cancer patients. A total of 364 luminal A, 175 luminal B, 239 HER2-enriched and 287 basal-like/claudin-low tumors were analyzed. (a) KLF4/5 were analyzed as single variables for all tumors combined. (b) KLF4/5 were analyzed as single variables within the basal-like/claudin-low and the HER2-enriched groups, as defined by PAM50 subtyping. (c) The outcome of patients harboring tumors with higher expression levels of both KLF4 and KLF5 (red, hi-hi) was compared with the outcome when tumors had lower expression levels of each factor (blue, lo-lo). For all curves, significance was determined using the log-rank (Mantel–Cox) test and P<0.05 was considered significant
Figure 3
Figure 3
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. 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
Figure 4
Figure 4
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
Figure 5
Figure 5
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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