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

Figures & data

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.

Table 1 Optimised Experimental protocols for PD0325901 MEKi Treatment.

Cell Line	Seeding Density	Length of Treatment (P < 0.05)	PD0325901 MEKi IC50 (Dose Curve R-Sq)	Versus Vehicle Control (DMSO)
N-Thy-Ori	1×10^5/ml	72Hrs	154.1uM (93%)	P < 0.01
8505c	1×10^5/ml	72Hrs	126.7uM (97.8%)	P < 0.01
SK-Mel-28	5×10^4/ml	48Hrs	1.28nM (86.0%)	P < 0.01
SK-Mel-31	5×10^4/ml	96Hrs	3.76nM (88.2%)	P < 0.01
SK-Mel-2	5×10^4ml	96Hrs	2.24nM (92.2%)	P < 0.01

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).

Table 2 Significant MIRNAs associated with samples.

8505C		NThy-Ori		SK-MEL-28		SK-MEL-31
Resistant (Fold Change >10)	Naïve (Fold Change <−10)	Resistant (Fold Change >10)	Naïve (Fold Change <−10)	Resistant (Fold Change >10)	Naïve (Fold Change <−10)	Resistant (Fold Change >10)	Naïve (Fold Change <−10)
MIR185MIR196bMIR199aMIR210MIR2110MIR218MIR223MIR2278MIR451MIR454MIR512MIR515^1MIR516aMIR516bMIR517aMIR517cMIR518bMIR518cMIR519aMIR519eMIR521MIR524MIR526bMIR874MIR1323	MIR211MIR130bMIR1910MIR200aMIR302aMIR373MIR579	MIR1-2	MIR19bMIR20aMIR29aMIR30aMIR32MIR33aMIR33bMIR96MIR99aMIR106bMIR148aMIR181dMIR183MIR188MIR195MIR210MIR215MIR299MIR301bMIR323MIR371MIR374aMIR376cMIR409MIR421MIR542MIR551aMIR620MIR652MIR708MIR941MIR1180MIR1254MIR1271MIR1296MIR1304MIR1977	MIR1274b	MIR26aMIR29bMIR30EMIR92aMIR99aMIR128MIR143MIR146aMIR151aMIR181bMIR211MIR218MIR302aMIR302bMIR302cMIR302dMIR339MIR378aMIR551b	MIR9-3MIR21MIR105-1MIR105-2MIR374bMIR1254	MIR143MIR302cMIR302dMIR371MIR454MIR592

¹ miRNAs in bold are members of the ESCC MIR302/373/374/520 family

Table 3 Association between top 20/cluster most significant GO terms.

GO Accession	Term	MIR520	MIR302
GO:0005515	protein binding	3.07E-13^1	1.67E-15
GO:0030154	cell differentiation	6.41E-11	1.11E-12
GO:0032502	developmental process	7.77E-31	7.66E-16
GO:0048518	positive regulation of biological process	2.37E-09	1.19E-14
GO:0048519	negative regulation of biological process	1.83E-20	7.16E-14
GO:0048522	positive regulation of cellular process	5.55E-10	1.67E-15
GO:0048523	negative regulation of cellular process	1.51E-19	3.69E-14
GO:0048869	cellular developmental process	6.41E-11	1.11E-12
GO:0050789	regulation of biological process	4.71E-21	1.16E-19
GO:0050794	regulation of cellular process	3.38E-23	2.06E-21
GO:0065007	biological regulation	4.71E-21	6.65E-21
GO:0043069	negative regulation of programmed cell death	6.79E-12	<0.01^2
GO:0006915	apoptotic process	1.27E-11	<0.01
GO:0012501	programmed cell death	1.27E-11	<0.01
GO:0042981	regulation of apoptotic process	1.27E-11	<0.01
GO:0043067	regulation of programmed cell death	1.34E-11	<0.01
GO:0008219	cell death	2.24E-11	<0.01
GO:0016265	death	2.24E-11	<0.01
GO:0048468	cell development	1.65E-10	<0.01
GO:0050896	response to stimulus	2.55E-07	<0.01
GO:0010467	gene expression	<0.01	1.56E-12
GO:0042127	regulation of cell proliferation	<0.01	1.11E-12
GO:0045449	regulation of transcription, DNA-dependent	<0.01	1.11E-12
GO:0019219	regulation of nucleobase-containing compound metabolic process	<0.01	9.32E-14
GO:0006355	regulation of transcription, DNA-dependent	<0.01	8.25E-13
GO:0019222	regulation of metabolic process	<0.01	1.34E-18
GO:0010468	regulation of gene expression	<0.01	1.93E-17
GO:0031323	regulation of cellular metabolic process	<0.01	6.29E-20
GO:0000307	cyclin-dependent protein kinase holoenzyme complex	–^3	3.46E-14

¹ Terms with the exact p-value stated were found in the top 20 terms for each group

² Terms with p-value <0.01 were significant but not within the top 20 most significant terms

³ Terms without a p-value were not significantly associated.

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).

Table 4 MIR302/373/374/520 embryonic stem cell specific cell cycle regulating (ESCC) microRNA family in holoclones compared to parental cells (relative fold change +/−2).

8505C		SK-MEL-28
miRNA	Fold Change	miRNA	Fold Change
MIR519A1	12.2	MIR302D	−8.10
517A	10.67	MIR302C	−7.09
MIR373	8.81	MIR302B	−6.54
MIR302A	5.61	MIR302A	−5.51
MIR302B	4.92	MIR516B2	−3.71
MIR302D	4.81	MIR520G	−3.71
MIR302C	4.38	MIR519D	−2.78

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.