Dysregulated JAK2 expression by TrkC promotes metastasis potential, and EMT program of metastatic breast cancer Min Soo Kim1, Joon Jeong2, Jeongbeob Seo3, Hae Suk Kim4, Seong-Jin Kim5#, and Wook Jin1,6#
1
Laboratory of Molecular Disease and Cell Regulation, Department of Biochemistry, School
of Medicine, Gachon University, Incheon 406-840, Korea 2
Department of Surgery, Gangnam Severance Hospital, Yonsei University Medical College,
712 Eonjuro, Gangnam-Gu, Seoul, 135-720, Korea 3
Medicinal Chemistry, CMG Pharma, 335, CHA Bio Complex, Pangyo-ro, Bundang-gu,
Seongnam-si, Gyeonggi-do, 13488, Korea, 4
TheragenEtex Bio Institute, TheragenEtex Co., Suwon, Gyeonggi-do 16229, Republic of
Korea 5
Nano-Bio Medicine Research Center, Advanced Institutes of Convergence Technology, and
Department of Transdisciplinary Studies, Graduate School of Convergence Science and Technology, Seoul National University, Suwon, Kyunggi-do 16229, Republic of Korea 6
#
Gachon Medical Research Institute, Gil Medical Center, Incheon, 405-760, Korea
To whom correspondence should be addressed. Email:
[email protected] or
[email protected]
Supplemental Figure Legend Figure S1. Increased expression of TrkC in human breast cancer and identification of TrkC knockdown. (a) The expression of TrkC mRNA in a panel of human normal, luminal, and basal-like cell lines was examined by TaqMan quantitative RT-PCR analysis. The endogenous 18S mRNA level was measured as an internal control. Data are presented as mean ± standard error of the mean (SEM). (b) RT-PCR and Western blot analysis of TrkC expression in SUM149 and Hs578T controls or TrkC-shRNA cells. GAPDH and β-actin were used as loading controls.
Figure S2. TrkC regulates STAT3 activity through induction of STAT3 expression. (a) Expression levels of mRNA encoding STAT3 in Hs578T control-shRNA or TrkC-shRNA cells. 18S mRNA was used to normalize variability in template loading. Data are presented as mean ± standard error of the mean (SEM). (b) Luciferase reporter assay of STAT3 in Hs578T controlshRNA or TrkC-shRNA cells. Each bar represents the mean ± SEM of three experiments. Data are presented as mean ± standard error of the mean (SEM).
Figure S3. Tyrosine kinase activity of TrkC is required for induction and stabilization of JAK2/STAT3/Twist-1. (a) Western blot analysis of the expression of the proteins P-JAK2, JAK2, P-STAT3, STAT3, and Twist-1 in SUM149 and Hs578T control-shRNA or TrkC-shRNA cells after treatment with or without MG132 (10 µg/ml) for 6 hr. β-actin was used as a loading control. (b) Immunoblot analysis of whole-cell lysates and immunoprecipitates derived from 293T cells transfected with the V5-TrkC (wild or kinase-dead mutants), HA-Ubiquitin, and Myc-Jak2 constructs as indicated. (c) Immunoblot analysis of whole-cell lysates and immunoprecipitates derived from 293T cells transfected with the V5-TrkC (wild or kinasedead mutants) and Myc-Jak2 constructs as indicated.
Figure S4. IL-6 neutralizing antibody suppresses TrkC-induced JAK2/STAT3 activity. Western blot analysis of the expression of the proteins P-JAK2, JAK2, P-STAT3, STAT3, and Twist-1 in SUM149 and Hs578T control-shRNA or TrkC-shRNA cells with or without treatment with IL-6 neutralizing antibody (5 ng/ml) for 8 hr. β-actin was used as a loading control.
Figure S5. c-Src leads to Twist-1 upregulation through activation of the JAK2/STAT3 pathway. (a) RT-PCR and Western blot analysis of c-Src expression in SUM149 and Hs578T controls or TrkC-shRNA cells. GAPDH and β-actin were used as loading controls. (b) Western blot analysis of the expression of the proteins P-JAK2, JAK2, P-STAT3, STAT3, and Twist-1 in Hs578T and SUM149 control-shRNA or c-Src-shRNA cells.
Figure S6. TrkC knockdown more effectively reduces Twist-1 and Twist-2 luciferase activity than SU6656. (a) Twist-1 or Twist-2 luciferase reporters were transfected into Hs578T cells expressing either control-shRNA or TrkC-shRNAs and then treated with 5µM SU6656 for 6 hr. Luciferase activity was measured 48 hr after transfection. Data are presented as mean ± standard error of the mean (SEM). (b) Twist-1 or Twist-2 luciferase reporters were transfected into SYF, SYF-Src, SYF-TrkC, or SYF-Src-TrkC cells, and luciferase activity was measured 48 hr after transfection. Data are presented as mean ± standard error of the mean (SEM).
Figure S7. TrkC upregulates Twist expression. (a) Relative Twist-1 or Twist-2 mRNA expression in Hs578T and SUM149 control-shRNA or TrkC-shRNA cells. The 18S mRNA was used to normalize variability in template loading. Data are presented as mean ± standard error of the mean (SEM). (b) Luciferase reporter assay of Twist-1 and Twist-2 in Hs578T and
SUM149 control-shRNA or TrkC-shRNA cells. Each bar represents the mean ± SEM of three experiments. Data are presented as mean ± standard error of the mean (SEM). (c) Twist-1 immunofluorescence staining in Hs578T control-shRNA or TrkC-shRNA cells. The red signal represents staining of Twist-1, while the blue signal represents DAPI nuclear DNA staining.
Figure S8. Contribution of TrkC to the metastatic ability of highly metastatic cancer cells. (a) Formation of spheroid colonies of SUM149 and Hs578T control-shRNA or TrkC-shRNA cells with or without 50 nM K252a treatment. Spheroid colonies in suspension were photographed at 200 × magnification. (b) Population doubling of SUM149 and Hs578T control-shRNA or TrkC-shRNA cells. Each data point represents the mean of cells counted in three dishes. Data are presented as mean ± standard error of the mean (SEM). (c) Soft agar colony-forming assay of SUM149 and Hs578T control-shRNA or TrkC-shRNA cells (n=3). *P
