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. 2016 May 17;7(20):29689-707.
doi: 10.18632/oncotarget.8822.

The transcription factor RUNX2 regulates receptor tyrosine kinase expression in melanoma

Rajeev K Boregowda  1 , Daniel J Medina  1 , Elke Markert  2 , Michael A Bryan  1 , Wenjin Chen  3   4 , Suzie Chen  5 , Anna Rabkin  5 , Michael J Vido  6 , Samuel I Gunderson  7 , Marina Chekmareva  4 , David J Foran  3   4 , Ahmed Lasfar  8   9 , James S Goydos  9 , Karine A Cohen-Solal  10
Affiliations

The transcription factor RUNX2 regulates receptor tyrosine kinase expression in melanoma

Rajeev K Boregowda et al. Oncotarget. .

Abstract

Receptor tyrosine kinases-based autocrine loops largely contribute to activate the MAPK and PI3K/AKT pathways in melanoma. However, the molecular mechanisms involved in generating these autocrine loops are still largely unknown. In the present study, we examine the role of the transcription factor RUNX2 in the regulation of receptor tyrosine kinase (RTK) expression in melanoma. We have demonstrated that RUNX2-deficient melanoma cells display a significant decrease in three receptor tyrosine kinases, EGFR, IGF-1R and PDGFRβ. In addition, we found co-expression of RUNX2 and another RTK, AXL, in both melanoma cells and melanoma patient samples. We observed a decrease in phosphoAKT2 (S474) and phosphoAKT (T308) levels when RUNX2 knock down resulted in significant RTK down regulation. Finally, we showed a dramatic up regulation of RUNX2 expression with concomitant up-regulation of EGFR, IGF-1R and AXL in melanoma cells resistant to the BRAF V600E inhibitor PLX4720. Taken together, our results strongly suggest that RUNX2 might be a key player in RTK-based autocrine loops and a mediator of resistance to BRAF V600E inhibitors involving RTK up regulation in melanoma.

Keywords: RUNX2; melanoma; receptor tyrosine kinase; resistance to targeted therapy; transcription factor.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1. RUNX2 knock down results in reduced expression of RTKs
A. Lysates from melanoma cell lines were analyzed for the expression of IGF-1R, FGFR1, PDGFRβ, EGFR and AXL. B. RUNX2 and RTKs levels in non-silencing ShRNA (NS), ShRUNX2-2 (Sh2) and ShRUNX2-3 (Sh3) stable melanoma cell lines. C. Levels of pAKT(T308), pAKT1(S473), pAKT2(S474), AKT, pERK1/2, ERK1/2 and Actin in non-silencing ShRNA (NS), ShRUNX2-2 (Sh2) and ShRUNX2-3 (Sh3) stable melanoma cell lines. D. Levels of RUNX2, FGFR1, EGFR, AXL, IGF-1R, PDFGRβ, phosphorylated EGFR (Y1068) and phosphorylated AXL (Y702) in C8161 and C81-61 melanoma cell lines.
Figure 2
Figure 2. RUNX2 and AXL co-expression in melanoma samples
Representative pictures of strong A., faint B. and negative C. AXL staining in RUNX2-expressing melanoma cores from a melanoma tissue microarray. RUNX2 staining (Chromogenic detection using red) in on the left panel, corresponding microscopic view of AXL staining (Chromogenic detection using DAB, brown) is on the right panel. D. Expression levels of RUNX2 and AXL in the 39 RUNX2-expressing melanoma samples analyzed. The Y axis shows quantitative Effective Staining Intensity (ESI; [71]) of RUNX2 in melanoma patient samples. The X axis expresses qualitative analysis of AXL staining defined as strong, faint and negative. The scale bar represents 50 μm.
Figure 3
Figure 3. U1 Adaptor oligonucleotides targeting RUNX2 inhibit RUNX2 and RTK expression
A. Expression of RUNX2 mRNA 72 hours after transfection of C8161 and 1205LU melanoma cell lines with the complexes, containing the dendrimer nanoparticle RGD-G5 and each of the 6 RUNX2 specific U1 Adaptors, named RA1, RA2, RA3, RA4, RA5 and RA6 (RA for RUNX2 Adaptor). This experiment, done in triplicate, is representative of three independent experiments. Results are expressed as % of relative RUNX2 mRNA normalized to the control NC3wt +/− SEM. B. Immunoblot analysis of RUNX2 expression 72 hours after transfection of C8161 and 1205LU melanoma cell lines with the complexes, containing the dendrimer nanoparticle RGD-G5 and two selected RUNX2 specific U1 Adaptors (RA2 and RA5 for C8161, RA2 and RA4 for 1205LU). C. Expression of FAK, IGF-1R and AXL 72 hours after transfection of C8161 cells with the RGD-G5-RA2 and RGD-G5-RA5 complexes and 72 hours after transfection of 1205LU cells with the RGD-G5-RA2 and RGD-G5-RA4 complexes. The samples were analyzed in quadruplicate. This experiment is representative of three independent experiments. Results are expressed as % of relative FAK, IGF1R or AXL mRNA normalized to the control NC3wt +/− SEM. * indicates p < 0.05, ** indicate p < 0.01 and *** indicate p < 0.001 compared with NC3wt based on Student's t-test.
Figure 4
Figure 4. AKT activity is involved in RUNX2 and RTK expression
1205LU A. and C8161 B. melanoma cell lines were treated with vehicle (DMSO, (D)) or 10 μM MK2206 (MK) for the indicated times. Lysates were analyzed for the expression of pAKT, AKT, RUNX2, EGFR, AXL and Actin.
Figure 5
Figure 5. Increased expression of RUNX2 isoforms and RTKs in melanoma cells resistant to BRAF V600E inhibition
A. An isoform level analysis of RUNX2 expression in 471 samples from the TCGA-SKCM (Skin Cutaneous Melanoma) cohort shows expression of variants 2 and 3 in the cohort, while variant 1 is mostly absent. Shown is the distribution of log-transformed expression values for each isoform (X axis) across the samples. Y axis: Number of samples. B. Expression of RUNX2 isoforms 1, 2 and 3 in melanoma cell lines. C. Clones resistant to 3 μM (clones 1-3), 5μM ((clone 4) and 0.5 μM (clones 5 and 6) PLX4720 were analyzed for RUNX2, EGFR, IGF-1R and AXL expression. P: Parental 1205LU melanoma cell line. D. Levels of pEGFR (Y1068) and pIGF-1R (Y1135/1136) in parental 1205LU melanoma cells (P) and clones 1, 4, 5 and 6 resistant to PLX4720. E. Levels of pERK1/2, pAKT (pan) (S473), pAKT (T308), ERK1/2 and AKT in clones 1, 4, 5 and 6 resistant to PLX4720.
Figure 6
Figure 6
A. Expression of RUNX2 and RTKs in melanoma cells rendered resistant to BRAF V600E inhibition in vivo. Melanoma cell lines established from PLX4720-resistant 1205LU xenografts (PRT lines 3, 4, 6, 9 and 11) were incubated in the presence of DMSO (D) or 1 μM PLX4720 (PLX) for 24 hours and RUNX2, AXL, IGF-1R and EGFR expression was analyzed. Low and high exposures for IGF1R are shown. B. Patient data from a cohort containing samples from untreated tumors and tumors treated with Vemurafenib and Dabrafenib respectively [56] were downloaded from GSE50509. Expression of RUNX2 transcripts 1, 2 and 3 in untreated and Vemurafenib and Dabrafenib treated groups. P-values were calculated using a t-test between untreated and vemurafenib treated groups.” C. 1205LU melanoma cells expressing non-silencing ShRNA (NS), ShRUNX2-2 (Sh2) or ShRUNX2-3 (Sh3) were treated with Vehicle (DMSO) or 10 μM PLX 4720 for 72 hours and viable cells were then counted. This experiment, done in triplicate, is representative of three independent experiments. Results are expressed as % (cell numbers in the presence of PLX4720/cell numbers in the presence of DMSO). *** indicate p < 0.001 compared with NS.
Figure 7
Figure 7. Working model for the role of RUNX2 in RTK-based autocrine loops and resistance to BRAF V600E targeted therapy
RUNX2 regulates RTK expression by mechanisms yet to be defined. Treatment of melanoma cells with BRAF V600E inhibitors (PLX4720, Vemurafenib) eventually results in up-regulation of RUNX2 and subsequent up-regulation of RTKs, implicated in resistance to BRAF V600E targeted therapy, through increased proliferation, survival, migration and invasion. Reactivation of the oncogenic MAPK pathway through RTK up-regulation compensates for the inhibition of BRAF V600E. Those RTKs also signal through the PI3K/AKT pathway and AKT activation contributes to maintain high RUNX2 levels. This positive feedback loop contributes to drive progression of resistant melanoma cells.
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