Abstract 4371: Transient receptor potential cation channel 3 (TRPC3) regulates tumor proliferation and migration of BRAF wild type human malignant melanoma
Authors: Oda Kayoko , Umemura Masanari , Katsumata Mayumi , Aoyama Haruki , Makino Ayako , Ohtake Makoto , Sato Itaru , Yamaguchi Yukie , Nagashima Yoji , Aihara Michiko , Ishikawa Yoshihiro , Nagasako Akane 
Published: August 2015

Background: It has been well known that melanoma has a poor prognosis due to its rapid progression and high metastatic ability before. Although a few new drugs came out into clinical field in very recent years, these drugs are restricted to use for the patients who have BRAF or MEK gene mutation. Therefore we still have to search new target points that are not dependent on these gene mutations. TRP channels are activated by changes of temperature or membrane voltage, resulting in activation of intracellular responses. Here, we investigate whether TRPC3 regulates cell proliferation and migration of human melanoma in vitro and in vivo.

Material: C8161 cells, a BRAF wild type human melanoma cell line, were mainly used in this study. Cell proliferation was assessed by 2,3, -bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt (XTT) assay. Cell cycle was analyzed by fluorescence-activated cells sorting. In order to examine the role of TRPC3 in human melanoma, Short heparin RNA (shRNA) transductions with lentivirus encoding either TRPC3 or scramble was performed according to the protocols of the manufacture. Secretion level of matrix metalloproteinase (MMP) 9 was assessed in the presence of Pyr3, TRPC selective inhibitor or not by gelatin zymography. We transplanted C8161 cells into the side chest of female Balb-c nude mice at the level of intradermal depth. Immediately after tumors were formed, Pyr3 or DMSO (control) was injected intradermaly around the tumor every day. We measured each diameters of tumor every two days, and calculated volume and regression rate.

Result: mRNA and protein of TRPC3 were expressed in multiple human melanoma cell lines and primary tissue. Proliferation rate of TRPC3 knocked down C8161 decreases in 48 hours (p&lt;0.0001). Pyr3, one of pyrazole compounds that is known to inhibit selectively TRPC3, suppressed cell proliferation (IC50 12.99 μM). Both knockdown of TRPC3 and Pyr3 decreased path length of migration (p&lt;0.01, p&lt;0.01 respectively). These results suggested that TRPC3 was involved in cell proliferation and migration of C8161. Pyr3 also shortened migration path length by tracking the movement of C8161cells. Secretion level of MMP9 was decreased by Pyr3. Pyr3 inhibited phosphorylation of signal transducer and activators of transcription (STAT) 5, suggesting that TRPC3-induced proliferation and migration were regulated by, at least in part, the JAK/STAT signaling pathway. Tumor volume transplanted on mice side chest was dramatically and immediately reduced by Pyr3 local injection, while that of control group was increased. These results indicated that TRPC3 is involved in cell proliferations not only in vivo but also in vitro.

Conclusion: TRPC3 regulates proliferation and migration, and thus tumor growth and metastasis of melanoma, suggesting that TRPC3 could be a novel target for treating BRAF wild type human melanomas.

Note: This abstract was not presented at the meeting.

Citation Format: Kayoko Oda, Masanari Umemura, Mayumi Katsumata, Haruki Aoyama, Ayako Makino, Makoto Ohtake, Itaru Sato, Yukie Yamaguchi, Yoji Nagashima, Michiko Aihara, Yoshihiro Ishikawa, Akane Nagasako. Transient receptor potential cation channel 3 (TRPC3) regulates tumor proliferation and migration of BRAF wild type human malignant melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4371. doi:10.1158/1538-7445.AM2015-4371