Lee Becky H, Neela Poornima H, Kent Michael S, Zehnder Ashley M
School of Veterinary Medicine, University of California-Davis, Davis, California, United States of America.
Program in Epithelial Biology, Stanford University, Stanford, California, United States of America.
PLoS One. 2017 Apr 26;12(4):e0176370. doi: 10.1371/journal.pone.0176370. eCollection 2017.
Canine oral mucosal melanoma is an aggressive malignant neoplasm and is characterized by local infiltration and a high metastatic potential. The disease progression is similar to that of human oral melanomas. Whereas human cutaneous melanoma is primarily driven by activating mutations in Braf (60%) or Nras (20%), human mucosal melanoma harbors these mutations much less frequently. This makes therapeutic targeting and research modeling of the oral form potentially different from that of the cutaneous form in humans. Similarly, research has found only rare Nras mutations and no activating Braf mutations in canine oral melanomas, but they are still reliant on MAPK signaling. IQGAP1 is a signaling scaffold that regulates oncogenic ERK1/2 MAPK signaling in human Ras- and Raf- driven cancers, including melanomas. To investigate whether IQGAP1 is a potential target in canine melanoma, we examined the expression and localization of IQGAP1 in primary canine melanomas and canine oral melanoma cell lines obtained from the University of California-Davis. Using CRISPR/Cas9 knockout of IQGAP1, we examined effects on downstream ERK1/2 pathway activity and assayed proliferation of cell lines when treated with a peptide that blocks the interaction between IQGAP1 and ERK1/2. We observed that canine IQGAP1 is expressed and localizes to a similar extent in both human and canine melanoma by qPCR, Western blot, and immunofluorescence. Deletion of IQGAP1 reduces MAPK pathway activation in cell lines, similar to effects seen in human BrafV600E cell lines. Additionally, we demonstrated reduced proliferation when these cells are treated with a blocking peptide in vitro.
犬口腔黏膜黑色素瘤是一种侵袭性恶性肿瘤，其特征是局部浸润和高转移潜能。该疾病的进展与人类口腔黑色素瘤相似。人类皮肤黑色素瘤主要由Braf（60%）或Nras（20%）的激活突变驱动，而人类黏膜黑色素瘤携带这些突变的频率要低得多。这使得口腔型黑色素瘤的治疗靶点和研究模型可能与人类皮肤型不同。同样，研究发现犬口腔黑色素瘤中只有罕见的Nras突变，没有激活的Braf突变，但它们仍然依赖MAPK信号传导。IQGAP1是一种信号支架，在包括黑色素瘤在内的人类Ras和Raf驱动的癌症中调节致癌性ERK1/2 MAPK信号传导。为了研究IQGAP1是否是犬黑色素瘤的潜在靶点，我们检测了IQGAP1在原发性犬黑色素瘤和从加利福尼亚大学戴维斯分校获得的犬口腔黑色素瘤细胞系中的表达和定位。使用CRISPR/Cas9敲除IQGAP1，我们检测了对下游ERK1/2通路活性的影响，并在用阻断IQGAP1和ERK1/2之间相互作用的肽处理细胞系时检测了细胞系的增殖情况。我们通过qPCR、蛋白质免疫印迹和免疫荧光观察到，犬IQGAP1在人类和犬黑色素瘤中的表达和定位程度相似。IQGAP1的缺失降低了细胞系中MAPK通路的激活，类似于在人类BrafV600E细胞系中观察到的效果。此外，我们证明了在体外使用阻断肽处理这些细胞时，细胞增殖减少。
