TRPM1 promotes tumor progression in acral melanoma by activating the Ca2+/CaMKIIδ/AKT pathway

Abstract

Introduction: Acral melanoma is a predominant and aggressive subtype of melanoma in non-Caucasian populations. There is a lack of genotype-driven therapies for over 50% of patients. TRPM1 (transient receptor potential melastatin 1), a nonspecific cation channel, is mainly expressed in retinal bipolar neurons and skin. Nonetheless, the function of TRPM1 in melanoma progression is poorly understood.

Objectives: We investigated the association between TRPM1 and acral melanoma progression and revealed the molecular mechanisms by which TRPM1 promotes tumor progression and malignancy.

Methods: TRPM1 expression and CaMKII phosphorylation in tumor specimens were tested by immunohistochemistry analysis and scored by two independent investigators. The functions of TRPM1 and CaMKII were assessed using loss-of-function and gain-of-function approaches and examined by western blotting, colony formation, cell migration and invasion, and xenograft tumor growth assays. The effects of a CaMKII inhibitor, KN93, were evaluated using both in vitro cell and in vivo xenograft mouse models.

Results: We revealed that TRPM1 protein expression was positively associated with tumor progression and shorter survival in patients with acral melanoma. TRPM1 promoted AKT activation and the colony formation, cell mobility, and xenograft tumor growth of melanoma cells. TRPM1 elevated cytosolic Ca²⁺ levels and activated CaMKIIδ (Ca²⁺/calmodulin-dependent protein kinase IIδ) to promote the CaMKIIδ/AKT interaction and AKT activation. The functions of TRPM1 in melanoma cells were suppressed by a CaMKII inhibitor, KN93. Significant upregulation of phospho-CaMKII levels in acral melanomas was related to increased expression of TRPM1. An acral melanoma cell line with high expression of TRPM1, CA11, was isolated from a patient to show the anti-tumor activity of KN93 in vitro and in vivo.

Conclusions: TRPM1 promotes tumor progression and malignancy in acral melanoma by activating the Ca^2+/CaMKIIδ/AKT pathway. CaMKII inhibition may be a potential therapeutic strategy for treating acral melanomas with high expression of TRPM1.

Keywords: Acral melanoma; Ca(2+) channel; CaMKII; TRPM1.

Graphical abstract

Fig. 1

TRPM1 was positively associated with tumor progression and poor survival in patients with acral melanoma. (A) Representative images of H&E staining and IHC analysis of TRPM1 expression in acral melanoma samples from patients. Magnified images of the boxed areas are shown. Scale bar: 100 μm. (B) TRPM1 expression versus the Breslow thickness of acral melanoma in the 87 patients. Breslow thickness < 2 mm, n = 31; Breslow thickness ≥ 2 mm, n = 56. (C) TRPM1 expression versus the T category of acral melanoma in the 87 patients. Tis, n = 6; T1, n = 14; T2, n = 16; T3, n = 18; T4, n = 33. (D) TRPM1 expression versus distant metastasis in the 87 patients. Patients with distal metastasis, n = 53; patients without distal metastasis, n = 34. (E) TRPM1 expression versus the AJCC stage in the 87 patients. Stage 0, n = 6; stage I, n = 17; stage II, n = 28; stage III, n = 17; stage IV, n = 19. The P values were determined by unpaired two-tailed Student’s t-test, *P < 0.05; **P < 0.01. Data are mean ± SEM. (F) Kaplan-Meier survival curves of 53 patients without distant metastasis. Patients were separated into a TRPM1-high group (H-scores ≥ 2, n = 19, red curve) and a TRPM1-low group (H-score < 2, n = 34, blue curve). The P values were determined by the log-rank test.

Fig. 2

TRPM1 activated AKT and promoted the growth, migration and invasion of melanoma cells and tumor growth. (A) Representative western blots of cells stably expressing either a scrambled shRNA or shRNAs specific for TRPM1. β -Actin served as a loading control, n = 3. (B) Representative images of clonogenic growth assays of cells stably expressing either a scrambled shRNA or TRPM1 shRNAs, n = 3. (C) Quantification of tumor growth curves for the mice bearing xenograft tumors derived from cells stably expressing either a scrambled shRNA or TRPM1 shRNA, n = 6. (D) Representative images of IHC analysis of TRPM1, phospho-AKT Thr308 (P-AKT) and Ki67 levels of xenograft tumor samples from the TRPM1 knockdown experiments, n = 6. (E) Representative western blots of cells stably carrying the empty vector or expressing 3xF-TRPM1, n = 3. (F) Quantification of clonogenic growth (n = 3), soft agar colony formation (n = 3), (G) cell migration (n = 3) and invasion (n = 4) assays in cells stably carrying the empty vector or expressing 3xF-TRPM1. (H) Quantification of tumor growth curves for the mice bearing xenograft tumors originating from cells stably carrying the empty vector or expressing 3xF-TRPM, n = 8. (I) Representative images of IHC analysis of TRPM1, P-AKT and Ki67 levels of xenograft tumor samples from the TRPM1 overexpression experiments, n = 8. Scale bar: 50 μm. The P values were determined by unpaired two-tailed Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001. Data are mean ± SEM.

Fig. 3

TRPM1 promoted cell growth and mobility by activating the Ca²⁺/CaMKIIδ/AKT pathway. (A) Quantification of cytosolic Ca²⁺ levels (upper) and representative western blots (bottom) in cells stably expressing either a scrambled shRNA or TRPM1 shRNAs (n = 3), or (B) in cells stably carrying the empty vector or expressing 3xF-TRPM1 (n = 3). (C) Representative western blots for CaMKIIδ or CaMKIIγ knockdown experiments on MMG1 cells stably carrying the empty vector or expressing 3xF-TRPM1, n = 3. (D) Representative western blots of CaMKIIδ coimmunoprecipitated with anti-AKT antibody-conjugated agarose in lysates from MMG1 cells stably carrying the empty vector or expressing 3XF-TRPM1. Mouse IgG antibodies served as an IP control, n = 3. (E) Representative western blots of F-CaMKIIδ coimmunoprecipitated with anti-AKT antibody-conjugated agarose in lysates from MMG1 cells stably carrying the empty vector or expressing F-CaMKIIδ constructs as indicated, n = 3. (F) Representative western blots of TRPM1 knockdown and 3xF-TRPM1 overexpression experiments on MMG1 cells stably carrying the empty vector or expressing F-CaMKIIδ constructs as indicated, n = 3. (G) Cell proliferation assays for CaMKIIδ knockdown experiments on MMG cells stably carrying the empty vector or expressing 3xF-TRPM1 at the indicated time points, n = 3. (H) Representative images of clonogenic growth and soft agar colony formation assays for CaMKIIδ knockdown experiments, n = 3. (I) Quantification of cell migration and invasion assays for CaMKIIδ knockdown experiments, n = 7. (J) Quantification of clonogenic growth for TRPM1 knockdown (left, n = 4) and 3xF-TRPM1 overexpression (right, n = 3) experiments on MMG1 cells stably carrying the empty vector or expressing F-CaMKIIδ constructs as indicated. P values were determined by unpaired two-tailed Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001. Data are mean ± SEM.

Fig. 4

CaMKII inactivation reduced the tumor-promoting activity of TRPM1. (A) Representative western blots of the KN93 treatment experiments on cells stably carrying the empty vector or expressing 3xF-TRPM1, n = 3. (B) Representative western blots of CaMKIIδ coimmunoprecipitated with anti-AKT antibody-conjugated agarose for the KN93 treatment experiments on MMG1 cells stably carrying the empty vector or expressing 3xF-TRPM1, n = 3. (C) Quantification of clonogenic growth, (D) soft agar colony formation, (E) migration, and (F) invasion assays for the KN93 treatment experiments on cells stably carrying the empty vector or expressing 3xF-TRPM1, n = 3. P values were determined by unpaired two-tailed Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001. Data are mean ± SEM.

Fig. 5

KN93 suppressed the growth of TRPM1-high acral melanoma. (A) Representative images of IHC analysis of phospho-CaMKII (P-CaMKII) levels in acral melanoma samples from patients. Magnified images of the boxed areas are presented. Scale bar: 100 μm. (B) The H-score of P-CaMKII versus the TRPM1 expression in acral melanoma samples from 27 patients in the KMUH cohort. Patients were separated into a TRPM1-low group (H-score < 1.0, n = 13) and a TRPM1-high group (H-score greater than 2.0, n = 14). (C) Representative western blots of various human melanoma cells. (D) Representative western blots of CA11 cells expressing either a scrambled shRNA or TRPM1 shRNAs, n = 3. (E) Quantification of tumor growth curves for the xenograft tumors originating from CA11 cells expressing either a scrambled shRNA or TRPM1 shRNA, n = 6. (F) Representative western blots of the KN93 treatment experiments on CA11 cells, n = 3. (G) Representative images of clonogenic growth and soft agar colony formation assays for the KN93 treatment experiments on CA11 cells, n = 3. (H) Quantification of cell migration and invasion assays for the KN93 treatment experiments on CA11 cells, n = 3. (I) Quantification of tumor growth curves for the mice bearing CA11 xenograft tumors treated with a mock agent (n = 8), 10 mg/kg KN93 three times weekly (3qw, n = 8), or five times weekly (5qw, n = 8) by intraperitoneal injection. (J) Representative images of H&E staining and IHC analysis of P-CaMKII, P-AKT, and Ki67 levels in CA11 xenograft tumor samples from the KN93 treatment experiments, Scale bar: 50 μm, n = 8. P values were determined by unpaired two-tailed Student’s t-test, *P < 0.05; **P < 0.001; ***P < 0.001. Data are mean ± SEM.