Pluripotency markers are differentially induced by MEK inhibition in thyroid and melanoma BRAFV600E cell lines

Abstract

Oncogenic mutations in BRAF are common in melanoma and thyroid carcinoma and drive constitutive activation of the MAPK pathway. Molecularly targeted therapies of this pathway improves survival compared to chemotherapy; however, responses tend to be short-lived as resistance invariably occursCell line models of melanoma and thyroid carcinoma, +/- BRAF(V600E) activating mutation, were treated with the MEK inhibitor PD0325901. Treated and naive samples were assayed for expression of key members of the MAPK pathway. Global microRNA expression profiling of naive and resistant cells was performed via next generation sequencingand indicated pluripotency pathways in resistance. Parental cell lines were progressed to holoclones to confirm the miRNA stemness profileMembers of the MIR302/373/374/520 family of embryonic stem cell specific cell cycle regulating (ESCC) microRNAs were identified as differentially expressed between resistant BRAF(V600E) melanoma and thyroid cell lines. Upregulated expression of gene and protein stemness markers, upregulated expression of MAPK pathway genes and downregulation of the ESCC MIR302 cluster in BRAF(V600E) melanoma indicated an increased stem-like phenotype in resistant BRAF(V600E) melanoma. Conversely, downregulated expression of gene and protein stemness markers, downregulated expression of MAPK pathway genes, upregulation of the ESCC MIR520 cluster, reeexpression of cell surface receptors, and induced differentiation-associated morphology in resistant BRAF(V600E) indicate a differentiated phenotype associated with MEK inhibitor resistance in BRAF(V600E) thyroid cellsThe differential patterns of resistance observed between BRAF(V600E) melanoma and thyroid cell lines may reflect tissue type or de novo differentiation, but could have significant impact on the response of primary and metastatic cells to MEK inhibitor treatment. This study provides a basis for the investigation of the cellular differentiation/self-renewal access and its role in resistance to MEK inhibition.

Keywords: BRAFV600E; MEK inhibition; cancer; cancer stem cell; microrna; pluripotency; resistance.

Figure 1.

BRAFT1799A. status of cell lines. Allelic discrimination plot for BRAFT1799A SNP. All samples were auto-called for BRAF status (95% Confidence Interval). X-axis: wild type allele; Y-axis: mutant allele.

Figure 2.

MEKi resistance induces differential gene expression of key members of the MAPK Pathway in BRAFV600E cancer. Cells treated with PD0325901 (resistant) or media-alone (naïve) were analyzed for gene expression. Fold change (RQ) expression in resistant samples was normalized to RQ of paired naïve samples and averaged across biological replicates. Statistical significance (*). Error bars: SEM. X-axis: Cell Line; Y-axis: Log2 (Avg RQ Resistant/RQ Naïve) Confocal analysis for tERK1/2 detects no change between Naïve and Resistant 8505C or NThy-Ori samples. pERK1/2 can be detected in all 3 layers of the cell in naïve samples whereas it is only detected in the apical and midsection in MEKi resistant 8505C samples. Conversely in NThy-Ori: pERK1/2 is only detected in the apical and midsection in naïve samples whereas pERK1/2 can be detected in all 3 layers in MEKi resistant samples. Red staining pERK1/2, green staining total ERK1/2, blue staining nuclear (DAPI).

Figure 3.

Holoclones have an upregulation of stemness factors. SK-MEL-28 and 8505C holoclones were compared to their parental cell lines for expression of pluripotency and EMT genes. RQ expression in holoclone samples was normalized to RQ of parental samples and averaged across biological replicates. Statistical significance (*). Error bars: SEM. X-axis: Assayed genes; Y-axis: Log2(Avg RQ Resistant/RQ Naïve).

Figure 4.

MEKi resistance induces upregulation of stemness-associated genes in BRAF^V600E melanoma cell lines. Cells treated with PD0325901 (resistant) or media-alone (naïve) were analyzed for gene expression. RQ expression in resistant samples was normalized to RQ of paired naïve samples and averaged across biological replicates. Statistical significance (*). Error bars: SEM. X-axis: Assayed cell line; Y-axis: Log₂(Avg RQ Resistant/RQ Naïve).

Figure 5.

Protein expression is differentially regulated in SK-MEL-28 and SK-MEL-2 MEKi resistant protein samples. (A-D) Western blot and autoradiograpgy intensity analysis was performed for PD0325901 resistant and naïve samples. X-axis: Cell Line; Y-axis: Intensity target protein/Intensity Actin.

Figure 6.

MEKi treatment induces reinstatement of TSHR protein in anaplastic thyroid cells (A) Gene expression analysis can detect TSHR expression in 8505C cells only following sustained MEK inhibitor treatment. TSHR is readily detectable in both the naïve and resistant BRAF^WT NThy-Ori cell line. (B) Confocal analysis detects expression of the TSHR protein expression in 8505C cells following sustained MEK inhibitor treatment. TSHR protein is readily detectable in both the naïve and resistant BRAF^WT NThy-Ori cell line. Confocal Legend: TSHR: Green; Actin: Red; Nucleus: Blue Images (individual color channels and merged) taken on 63X objective lens.