Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 26 (5), 738-53

TRPM3 and miR-204 Establish a Regulatory Circuit That Controls Oncogenic Autophagy in Clear Cell Renal Cell Carcinoma

Affiliations

TRPM3 and miR-204 Establish a Regulatory Circuit That Controls Oncogenic Autophagy in Clear Cell Renal Cell Carcinoma

Daniel P Hall et al. Cancer Cell.

Abstract

Autophagy promotes tumor growth by generating nutrients from the degradation of intracellular structures. Here we establish, using shRNAs, a dominant-negative mutant, and a pharmacologic inhibitor, mefenamic acid (MFA), that the Transient Receptor Potential Melastatin 3 (TRPM3) channel promotes the growth of clear cell renal cell carcinoma (ccRCC) and stimulates MAP1LC3A (LC3A) and MAP1LC3B (LC3B) autophagy. Increased expression of TRPM3 in RCC leads to Ca(2+) influx, activation of CAMKK2, AMPK, and ULK1, and phagophore formation. In addition, TRPM3 Ca(2+) and Zn(2+) fluxes inhibit miR-214, which directly targets LC3A and LC3B. The von Hippel-Lindau tumor suppressor (VHL) represses TRPM3 directly through miR-204 and indirectly through another miR-204 target, Caveolin 1 (CAV1).

Figures

Figure 1
Figure 1. TRPM3 channel is overexpressed in ccRCC and regulates growth of xenograft tumors
(A) Box-whisker plot showing quantification of the tumor/kidney ratio of TRPM3 protein levels in tumors with wild-type (WT) or inactive VHL. The boxes represent lower and upper quartiles separated by the median (thick horizontal line) and the whiskers extend to the minimum and maximum values. Means ± SD of each distribution are indicated by closed dots and crosses on the whiskers, respectively. (B) Representative western blot showing expression of TRPM3 protein in lysates of ccRCCs (T) with deleted VHL compared to adjacent kidneys (K). Different bands detected by the antibody may correspond to different splice variants of TRPM3 (Ensembl) or post-translational modifications. (C) Representative images of TRPM3 immunocytochemistry in sections of two different ccRCCs and normal kidney. (D) Bar graph showing incidence and weight of tumors formed in orthotopic xenografts by VHL(−) cells with endogenous (endog.) TRPM3, TRPM3KD (shRNA I and III) and TRPM3DN. Tumor weight includes kidney in which the tumor was growing. NK-average weight of normal kidney. (E) H&E staining of representative sections of xenograft tumors formed by 786-O VHL(−) cells shown in 1D. Clear separation of tumors from kidneys in the case of tumors formed by cells with TRPM3KD(shIII) or TRPM3DN is indicated by black dashed line. (F) Representative sections from tumors formed by 786-O VHL(−) cells shown in panel 1D stained for Ki67 and bar graph quantification of Ki67 positive nuclei compared to the total number of nuclei. (G) Box-whisker plot showing subcutaneous growth of tumors formed by 786-O VHL(−) cells with endogenous TRPM3 or TRPM3R cells. Open circles represent points that are outside the 1.5 interquartile range from the box. Day 1 corresponds to the time point 2.5 weeks after injections. (H) Examples and incidence of tumors formed by 786-O VHL(−) cells lines with indicated status of TRPM3. Scale bar = 1 cm. (I) Weight of the collected tumors formed by 786-O VHL(−) cells lines with indicated status of TRPM3. (J) Representative H&E staining of sections from tumors shown in Figure 1H. (K) Representative sections from subcutaneous tumors in Figure 1H stained for Ki67 and bar graph quantification of Ki67 positive nuclei compared to the total number of nuclei. Unless otherwise indicated, all scale bars = 50μm, and error bars indicate SEM. See also Figure S1.
Figure 2
Figure 2. VHL inhibits TRPM3 expression through direct targeting by miR-204
(A) Western blot showing levels of TRPM3 expression in VHL(−) cells and in Caki-1 cell line with endogenous VHL. (B) Western blot showing TRPM3 levels in 786-O and A498 cells with reconstituted VHL (VHL(+)), compared to the isogenic VHL(−) cells. (C) Western blot showing effects of anti-miR-204 on expression of TRPM3 protein in the indicated VHL(+) RCC cells. (D) Western blot showing effects of pre-miR-204 on expression of TRPM3 in the indicated VHL(−) RCC cells. (E) Effects of miR-204 on activity of the luciferase reporter construct containing WT or mutated in miR-204 binding site (mut) 3′UTR of TRPM3 in 786-O VHL(−) cells. The ratio of luciferase activity in cells transduced with wild-type miR-204 to activity in cells transduced with non-targeting lentivirus control is shown. Error bars indicate SEM. (F) Representative western blot showing expression of TRPM3 protein (top) in nine kidneys with low and high levels of miR-204 (bottom). (G) Regression analysis showing negative correlation between normalized TRPM3 protein level and normalized miR-204 level in human kidneys. Each dot represents a sample and the solid line represents the linear regression fit, with the Pearson correlation coefficient (r) as well as the slope and intercept of the fitted line shown in the upper left corner of the box. See also Figure S2.
Figure 3
Figure 3. CAV1 is a miR-204 target and regulates expression of TRPM3
(A) Western blot showing levels of CAV1 expression in indicated cell lines. (B) Expression of CAV1 protein and mRNA using Western blot (left) and QRT-PCR (right) in 786-O and A498 VHL(+) cells compared to the isogenic VHL(−) cells. Error bars indicate SD. (C) Western blot showing effects of pre-miR-204 on expression of CAV1 protein in VHL(−) cells. (D) Western blot showing effects of anti-miR-204 on expression of CAV1 protein in VHL(+) cells. (E) Effects of miR-204 on the activity of a luciferase reporter containing WT or mutated in miR-204 binding site (mut) chimeric 3′UTR of CAV1 in 786-O VHL(−) cells. Error bars indicate SEM. (F) Effects of CAV1KD on expression levels of TRPM3 protein in 786-O VHL(−) cells. (G) Effects of stable overexpression of Flag-tagged CAV1 on TRPM3 protein levels in two different pools (P1 and P2) of 786-O VHL(+) cells. Endogenous CAV1-endog. CAV1 (H) Regression analysis demonstrating positive correlation between normalized TRPM3 and normalized CAV1 protein levels in human ccRCCs. Analysis was performed as in Figure 2G. (I) Representative western blot showing levels of TRPM3 and CAV1 in 14 human ccRCCs. See also Figure S3.
Figure 4
Figure 4. TRPM3 channel regulates autophagy
(A) Bar graph quantification of the number of autophagic vesicles (top) and the cytoplasmic area (bottom) in TEM sections of 20 786-O VHL(−) control (stably transfected with scrambled pLKO.1 vector (Scr)) or TRPM3KD cells. In this and all subsequent figures, if not otherwise indicated, term “TRPM3KD” refers to knockdowns using shRNA I. (B) Western blot and bar graph quantification showing effects of TRPM3KD on accumulation of LC3A/B-II in VHL(−) cells. Dashed line shows levels of LC3-II accumulation in control cells which is accepted as 1. (C) Western blots showing effects of TRPM3DN on the accumulation of LC3A/B-II in 786-O VHL(−) cells. P1 and P2 are two separate pools of cells. (D) Western blot showing rescue of LC3A/B-II accumulation upon reconstitution of TRPM3R in 786-0 VHL(−) TRPM3KD cells. (E) Western blot showing effects of TRPM3KD on accumulation of LC3A/B-II in VHL(+) cells. (F) Immunostaining for LC3B puncta in sections from respective orthotopic xenografts of 786-O VHL(−) cells with indicated status of TRPM3. Scale bar = 20 μm. (G) Immunostaining for LC3B puncta in sections from subcutaneous xenografts of 786-O VHL(−) cells with indicated status of TRPM3. Scale bar as in Figure 4F. (H) Incidence and weight of tumors formed in orthotopic xenografts by 786-0 VHL(−) TRPM3KD cells with re-expressed Flag-LC3B. Note that TRPM3KD(shI) cells did not form tumors, similarly as shown in Figure 1D. In all panels error bars indicate SEM. See also Figure S4.
Figure 5
Figure 5. TRPM3 regulates autophagy through CAMKK2/AMPK/ULK1 pathway
(A) Bar graph quantification of ATG-16L puncta detected by immunofluorescence staining in 786-O VHL(−) control and TRPM3KD cells. (B) Bar graph quantification of ATG5-ATG12 puncta detected by immunofluorescence staining in 786-O VHL(−) control and TRPM3KD cells. In panels A and B error bars indicate SEM. (C) Western blot showing steady-state expression levels of ATG16L protein and ATG5-ATG12 conjugate in 786-O VHL(−) control and TRPM3KD cells. (D) Western blot showing effects of TRPM3KD on expression and phosphorylation of AMPKα and ULK1 in VHL(−) cells. (E) Western blot showing effects of TRPM3R on expression and phosphorylation of AMPKα andULK1 in 786-O VHL(−) TRPM3KD cells. (F) Western blot showing effects of TRPM3KD on expression and phosphorylation of AMPKα and ULK1 in VHL(+) cells. (G) Western blot showing effects of the constitutively active CAMKK2 on accumulation of LC3A/B-II and phosphorylation of AMPKα and ULK1 in 786-O VHL(−) TRPM3KD cells. EV-empty virus. (H) Western blot showing effects of CAMKK2 shRNA on accumulation of LC3A/B-II, and phosphorylation of AMPK and ULK1 in 786-O VHL(−) cells. (I) Western blot showing effects of the constitutively active AMPKα2 on accumulation of LC3A/B-II, and phosphorylation of AMPK and ULK1 in 786-O TRPM3KD cells. EV-empty virus. (J) Western blot showing effects of AMPKα1/α2 shRNAs on accumulation of LC3A/B-II, and phosphorylation of AMPK and ULK1 in 786-O VHL(−) cells. See also Figure S5.
Figure 6
Figure 6. TRPM3 regulates autophagy through miR-214
(A) Normalized levels of miR-214 in 786-O VHL(−) control, TRPM3KD and TRPM3R cells. (B) Normalized levels of miR-214 in 786-O VHL(−) control, TRPM3KD, and VHL(+) cells. (C) Normalized levels of miR-214 in VHL(−) RCC cell lines and VHL(+) Caki-1 cell line. (D) Effects of overexpression of TRPM3WT or TRPM3DN on the steady-state levels of miR-214 in indicated cells. (E) Effects of TRPM3KD on normalized levels of miR-214 in VHL(+) cells. (F) Effects of treatment with the indicated concentrations of pre-miR-214 and anti-miR-214 on accumulation of LC3A/B in starved VHL(−) cells. All cells were treated with CQ. (G) Effects of anti-miR-214 (lentivirus) treatments on the accumulation of LC3A/B in starved 786-O VHL(−) TRPM3KD cells. In the case of siRNA, only CQ treated cells are shown. (H) Effects of lentiviral anti-miR-214 and pre-miR-214 treatments on the accumulation of LC3A/B in starved 786-O VHL(+) cells. (I) Effects of pre-miR-214 on the activity of luciferase reporter constructs containing LC3A or LC3B 3′UTRs with either WT or mutated (mut) miR-214 binding sites in 786-O VHL(−) cells. (J) Effects of constitutively active CAMKK2 and AMPKα2 on the accumulation of miR-214 in starved 786-O VHL(−) TRPM3KD cells. (K) Effects of CAMKK2, AMPKα1/2, ULK1 and PIK3C3 knockdowns on the accumulation of miR-214 in starved 786-O VHL(−) cells. (L) Effects of anti-miR214 on the accumulation of LC3A and LC3B in 786-O VHL(−) cells treated with STO-609. In all panels n represents a number of individual experiments in triplicate. If n≥3, error bars indicte SD; if n≥3, error bars indicate SEM. See also Figure S6.
Figure 7
Figure 7. Effects of TRPM3KD are reproduced by MFA, a small molecule blocker of TRPM3
(A) Box-whisker plot showing growth of subcutaneous xenograft tumors formed by indicated VHL(−) cells in mice treated with MFA, IDM or vehicle. Open circles represent points that are outside the 1.5 interquartile range from the box. (B) Representative sections of 786-O tumors from each treatment group stained for Ki67 and bar graph quantification performed as in Figure 1F. Scale bar = 100 μM. (C) Effects of treatment with MFA on expression of endogenous TRPM3 measured by western blot and by QRT-PCR in VHL(−) cells. (D) Effects of MFA treatments of 786-O and A498 VHL(−) cells on accumulation of LC3A/B-II and phosphorylation of ULK1-Ser317. R: ratio of p-ULK1-Ser317 to total ULK1. (E) Effects of MFA on accumulation of miR-214 in 786-O and A498 VHL(−) cells. In all panels error bars indicate SEM. See also Figure S7.
Figure 8
Figure 8. Model summarizing proposed regulatory network controlling oncogenic LC3A/LC3B autophagy
Black lines represent oncogenic pathways; gray lines represent tumor suppressing pathways. * work reported by Mikhaylova et al., 2012.

Comment in

  • Targeting Ions-Induced Autophagy in Cancer
    F Cecconi et al. Cancer Cell 26 (5), 599-600. PMID 25517744.
    Autophagy is an important cellular homeostasis pathway, but its role in cancer remains to be fully elucidated. In this issue of Cancer Cell, Hall and colleagues describe …

Similar articles

See all similar articles

Cited by 43 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback