Dysregulated JAK2 expression by TrkC promotes metastasis potential, and EMT program of metastatic breast cancer

Abstract

Metastatic breast cancers are aggressive tumors associated with high levels of epithelial-mesenchymal transition (EMT) markers, activation of IL6/JAK2/STAT3 and PI3K/AKT pathways for cell growth, mobility, invasion, metastasis, and CSC status. We identified a new molecular and functional network present in metastasis that regulates and coordinates with TrkC. Inhibition of SOCS3-mediated JAK2 degradation by TrkC increases total JAK2/STAT3 expression, and then leads to upregulation of Twist-1 through activation of JAK2/STAT3 cascade. Also, TrkC increases secretion and expression of IL-6, suggesting that this autocrine loop generated by TrkC maintains the mesenchymal state by continued activation of the JAK2/STAT3 cascade and upregulation of Twist expression. Moreover, TrkC interacts with the c-Src/Jak2 complex, which increases Twist-1 and Twist-2 levels via regulation of JAK2/STAT3 activation and JAK2/STAT3 expression. Furthermore, TrkC enhances metastatic potential of breast cancer via induction of EMT by upregulating Twist-1 and Twist-2. Additionally, TrkC significantly enhances the ability of breast cancer cells to form pulmonary metastases and primary tumor formation. Unexpectedly, we found that TrkC expression and clinical breast tumor pathological phenotypes show significant correlation. These findings suggest that TrkC plays a central role in tumorigenicity, metastasis, and self-renewal traits of metastatic breast cancer.

Figure 1. Increased expression of TrkC in human breast carcinomas.

(a) Relative levels of TrkC expression in normal or invasive breast carcinoma samples of 17 individual breast cancer patients. The expression was compared to that of healthy tissue, and the endogenous 18S mRNA level was measured as an internal control. A P value was considered to be significant in t-test. (b) The gene expression data were plotted as Box-and-whisker (Tukey) plots of mean TrkC expression levels in molecular subtypes of breast cancer patients. The signatures of TrkC level extracted from 2,136 Curtis and 473 UNC datasets and averaged for each tumor. Points below and above the whiskers are drawn as individual dots. A P value was considered to be significant in ANOVA. (c) Relative TrkC mRNA expression in seven individual normal human tissues or triple-negative breast carcinoma samples. The endogenous 18S mRNA level was measured as an internal control. A P value was considered to be significant in t-test. (d) The mean TrkC expression in other subtypes or triple-negative subtypes of human breast cancer patients from three publicly available data sets (Curtis, Gluck, and UNC datasets). The TrkC gene expression level was extracted from the dataset and averaged for each tumor. Points below and above the whiskers are drawn as individual dots. A P value was considered to be significant in t-test. (e) Pattern of TrkC expression in a series of 59 breast cancer samples. Representative immunohistochemical images of TrkC staining in normal human breast tissue, infiltrating duct carcinoma, and metastatic carcinoma in the lymph nodes (magnification: 200×). (f) Patients were divided into those who expressed high and low TrkB levels, and their survival rates from a publicly available data set (UNC) were compared. The P value was calculated by a log-rank test.

Figure 2. TrkC regulates JAK2/STAT3 activity and directly regulates its downstream target.

(a) Western blot analysis of the expression of P-JAK2, JAK2, P-STAT3, STAT3, and Twist-1 proteins in Hs578T and SUM149 cells with or without 50 nM K252a treatment for 16 hr. β-actin was used as a loading control. (b) Western blot analysis of the expression of P-JAK2, JAK2, P-STAT3, STAT3, and Twist-1 proteins in Hs578T and SUM149 cells with or without 15μM AG490 (JAK2 inhibitor) treatment for 16 hr. β-actin was used as a loading control. (c) Western blot analysis of the expression of P-JAK2, JAK2, P-STAT3, STAT3, and Twist-1 proteins in MCF10A or MCF10A-TrkC cells with or without 50 nM K252a treatment for 16 hr. β-actin was used as a loading control. (d) Western blot analysis of the expression of P-JAK2, JAK2, P-STAT3, STAT3, and Twist-1 proteins in Hs578T and SUM149 control-shRNA or TrkC-shRNA cells. β-actin was used as a loading control. (e) Western blot analysis of the expression of P-JAK2, JAK2, P-STAT3, STAT3, and Twist-1 proteins in MCF10A, MCF10A-TrkC, or MCF10A-TrkC kinase-dead mutant (K572N, Yx3F) cells. β-actin was used as a loading control. (f) 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 transfection of TrkC or TrkC kinase-dead mutant (K572N, Yx3F) constructs.

Figure 3. TrkC induces JAK2 stabilization by inhibiting SOCS3-mediated degradation of JAK2.

(a) RT-PCR and western blot analysis of JAK2 expression in Hs578T and SUM149 control-shRNA or TrkC-shRNA cells. GAPDH and β-actin were used as loading controls. (b) Immunoblot analysis of whole-cell lysates and immunoprecipitates derived from 293T cells transfected with V5-TrkC, HA-Ubiquitin, and Myc-Jak2 constructs as indicated. (c) Immunoblot analysis of whole-cell lysates and immunoprecipitates derived from 293T cells transfected with V5-TrkC, HA-Ubiquitin, Flag-SOCS3, and Myc-Jak2 constructs as indicated. (d) TrkC interacts with JAK2. Immunoblot analysis of whole-cell lysates and immunoprecipitates derived from 293T cells transfected with the V5-TrkC and Myc-Jak2 constructs as indicated. (e) Identification of the JAK2 region responsible for TrkC interaction and immunoblot analysis of whole-cell lysates and immunoprecipitates derived from 293T cells transfected with the V5-TrkC and Myc-Jak2 deletion constructs as indicated. (f) Identification of the inhibition of SOCS3-JAK2 complex formation by TrkC and immunoblot analysis of whole-cell lysates and immunoprecipitates derived from 293T cells transfected with the V5-TrkC, Flag-SOCS3, and Myc-Jak2 constructs as indicated. (g) Identification of endogenous SOCS3-JAK2 complexes in Hs578T and SUM149 control-shRNA or TrkC-shRNA cells.

Figure 4. Increased secretion of IL-6 by TrkC correlates with increased nuclear translocation of STAT3.

(a) ELISA assay of IL-6 secretion by Hs578T control-shRNA or TrkC-shRNA cells (n = 3). Data are presented as mean ± standard error of the mean (SEM). (b) Expression levels of mRNA encoding IL-6 in basal-like breast cancer cells (MDA-MB-231, MDA-MB-435, Hs578T, and SUM149) and normal or nontransformed cells (HMLE, MCF7, or MCF10A breast cells). 18S mRNA was used to normalize variability in template loading. Data are presented as mean ± standard error of the mean (SEM). (c) Expression levels of mRNA encoding IL-6 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). (d) Western blot analysis of the expression of P-JAK2, JAK2, P-STAT3, STAT3, and Twist-1 proteins in Hs578T and SUM149 control-shRNA or TrkC-shRNA cells with or without IL-6 treatment. β-actin was used as a loading control. (e) Immunofluorescence staining of the nuclear translocation of STAT3 in Hs578T and SUM149 control-shRNA or TrkC-shRNA cells with or without IL-6 treatment. The green signal represents staining of the corresponding protein, while the blue signal represents DAPI nuclear DNA staining. (f) Expression levels of mRNA encoding Twist-1 and Twist-2 in Hs578T control-shRNA or TrkC-shRNA cells with or without IL-6 treatment. 18S mRNA was used to normalize variability in template loading. Data are presented as mean ± standard error of the mean (SEM). (g) Luciferase reporter assay of Twist-1 and Twist-2 in Hs578T control-shRNA or TrkC-shRNA cells with or without IL-6 treatment. Each bar represents the mean ± SEM of three experiments. Data are presented as mean ± standard error of the mean (SEM). Some of the bar graphs do not have visible error bars due to low values of standard error of the mean (SEM).

Figure 5. TrkC/c-Src or TrkC interacts with JAK2 and leads to Twist-1 upregulation through activation of the JAK/STAT3 pathway.

(a) Western blot analysis of the expression of P-JAK2, JAK2, P-STAT3, STAT3, and Twist-1 proteins in Hs578T and SUM149 cells with or without 5 μM SU6656 treatment for 6 hr. β-actin was used as a loading control. (b) TrkC or TrkC/c-Src complex interacts with JAK2. Immunoblot analysis of whole-cell lysates and immunoprecipitates derived from 293T cells transfected with the V5-TrkC, HA c-Src, and Myc-Jak2 constructs as indicated. (c) Identification of complex formation of endogenous TrkC/c-Src/JAK2 in Hs578T and SUM149 cells. (d) TrkC interacts with JAK2 in SYF-TrkC cells or SYF-Src-TrkC cells after transfection with Myc-Jak2. (e) Activation and expression of Jak2 in SYF cells or SYF-TrkC cells after immunoprecipitation was examined by immunoblotting using the indicated antibodies. (f) Western blot analysis of the expression of P-JAK2, JAK2, P-STAT3, STAT3, and Twist-1 proteins in SYF cells or SYF-TrkC cells. (g) Western blot analysis of expression of P-JAK2, JAK2, P-STAT3, STAT3, and Twist-1 proteins in SYF, SYF-TrkC, SYF-cSrc, and SYF-cSrc-TrkC cells. (h) Expression levels of mRNA encoding Twist-1 and Twist-2 in Hs578T control-shRNA or TrkC-shRNA cells with or without 5 μM SU6656 treatment for 6 hr. 18S mRNA was used to normalize variability in template loading. Data are presented as mean ± standard error of the mean (SEM).

Figure 6. TrkC induces the EMT program by upregulating Twist.

(a) Western blot analysis of the expression of TrkC, E-cadherin, α-catenin, β-catenin, N-cadherin, and fibronectin proteins in MCF10A and MDCK control or TrkC cells. β-actin was used as a loading control. (b) The expression levels of mRNA encoding E-cadherin and N-cadherin in MDCK or MDCK-TrkC cells. 18S mRNA was used to normalize variability in template loading. Data are presented as mean ± standard error of the mean (SEM). (c) Luciferase reporter assay of E-cadherin in MDCK or MDCK-TrkC cells. Each bar represents the mean ± SEM of three experiments. Data are presented as mean ± standard error of the mean (SEM). (d) Immunofluorescence staining of E-cadherin and N-cadherin in MDCK or MDCK-TrkC cells. The red signal represents the staining of the corresponding protein, while the blue signal represents DAPI nuclear DNA staining. (e,f) RT-PCR analysis of E12, Goosecoid, SIP1, or Slug mRNA levels (f) and immunoblotting analysis of Twist-1 levels (f) in MDCK or MDCK-TrkC cells. β-actin and GAPDH were used as loading controls. (g) Luciferase reporter assay of Twist-1 and Twist-2 in MDCK or MDCK-TrkC cells. Each bar represents the mean ± SEM of three experiments. Data are presented as mean ± standard error of the mean (SEM). Some of the bar graphs do not have visible error bars due to low values of standard error of the mean (SEM).

Figure 7. Suppression of TrkC expression inhibited the ability of Hs578T cells to metastasize from the mammary gland to the lung.

(a) Volumes of primary mammary tumors formed by Hs578T control-shRNA or TrkC-shRNA cells. Each data point represents the mean of each type of primary tumor. A P < 0.0001 was considered to indicate significance for ANOVA. (b) Representative images of tumors from mice harboring Hs578T control-shRNA or TrkC-shRNA mammary tumors after tumor implantation. (c) Volumes of primary mammary tumors formed by MCF10A-Ras or MCF10A-Ras-TrkC cells. Each data point represents the mean of each type of primary tumor. A P < 0.0001 was considered to indicate significance for ANOVA. (d) Representative images of tumors from mice harboring MCF10A-Ras or MCF10A-Ras-TrkC cells. (e) LOXO-101 inhibits tumor growth in a Hs578T xenograft model. 1 × 10⁶ cells were implanted into the mammary fat pads of mice and treated with diluent (control) or LOXO-101 (60 mg/kg/dose) twice daily for 14 days when the tumor size was 200 ± 20 mm³. (f) Representative images of tumors from mice harboring diluent (control) or LOXO-101 (60 mg/kg/dose) (g) Total number of lung metastatic nodules in each mouse in each group were counted using a dissection scope. Two populations of Hs578T cells that were independently infected with TrkC-shRNA #1 and TrkC-shRNA #2 were used in independent experiments (n = 6) and consistently yielded similar results. A P < 0.0001 was considered to indicate significance for ANOVA. (h) Representative images and H&E staining sections of mouse lung lobes 35 days after tail vein injection of Hs578T control-shRNA or TrkC-shRNA cells. N, Normal lung tissue; M, metastatic nodule. (i) Immunoblotting of the expression of TrkC and Twist-1 in tumor cells recovered from the lungs of individual mice expressing either Hs578T control-shRNA or TrkC-shRNA. Some of the bar graphs do not have visible error bars due to low values of standard error of the mean (SEM).