Transient receptor potential melastatin 6 and transient receptor potential melastatin 6/7 antagonists suppress colon adenocarcinoma HT-29 cells

Abstract

BACKGROUND

Magnesium (Mg²⁺) plays a fundamental role in numerous cellular processes, including enzymatic reactions, DNA replication, oxidative stress response, and cytoskeletal dynamics. In fact, dysregulation of Mg²⁺ homeostasis has been increasingly associated with the development and progression of cancer, particularly colorectal cancer (CRC). Transient receptor potential melastatin (TRPM) channels, especially TRPM6 and TRPM7, are essential regulators of epithelial Mg²⁺ influx. While TRPM7 promotes CRC progression, the role of TRPM6 and TRPM6/7 channels remains unclear.

AIM

To investigate the role of membrane-localized TRPM6 and TRPM6/7 channels in Mg²⁺ influx, spheroid (SP) formation, stemness, and migration.

METHODS

We used parental and SP-derived HT-29 cells at comparable passages as in vitro models. Mass spectrometry confirmed full-length sequences, phosphorylation, and methionine oxidation of TRPM6 and TRPM7. Mg²⁺ influx, total and free Mg²⁺ levels were measured by fluorescence imaging and biochemical assays. TRPM6 / TRPM7 expression and markers were analyzed by western blot. Functional assays, including secondary SP formation and wound healing, assessed stemness and migration. Cells were treated with Mg²⁺ transport inhibitors: Co(III)hexamine, 2-aminoethyl diphenylborinate (TRPM6/7 blocker), and Mesendogen (TRPM6 inhibitor).

RESULTS

The expression of membrane-bound TRPM6, TRPM7, and TRPM6/7 was significantly higher in SP cells than in parental cells. Mass spectrometric analysis confirmed the presence of full-length TRPM6 and TRPM7 with increased phosphorylation and oxidation in SP cells. Enhanced Mg²⁺ influx and total intracellular Mg²⁺ levels were observed in SP cells. Free ionized intracellular Mg²⁺ levels remained comparable across all experimental groups. Pharmacological inhibition of TRPM6 and TRPM6/7 significantly reduced Mg²⁺ influx, decreased total Mg²⁺ content, compromised CRC SP stability, abolished cancer stem-like properties, impaired cell migration, and downregulated pro-tumorigenic markers, including Nanog, cyclooxygenase-2, and matrix metalloproteinase-9.

CONCLUSION

Membrane-localized TRPM6 and TRPM6/7 channels regulate Mg²⁺ influx and promote CRC stemness, SP stability, and migration, highlighting their potential as therapeutic targets to inhibit CRC progression and metastasis.

Key Words: Cancer stem cells; Cellular Mg²⁺ content; Colorectal cancer; Transient receptor potential melastatin 6/7; Transient receptor potential melastatin 6

Core Tip: This study demonstrated that colorectal cancer (CRC) spheroids (SPs) exhibited significantly increased membrane expression and phosphorylation of transient receptor potential melastatin (TRPM) 6 and TRPM6/7 channels, which enhanced magnesium (Mg²⁺) influx and promoted intracellular Mg²⁺ accumulation. However, selective TRPM6 and TRPM6/7 inhibitors markedly suppressed Mg²⁺ influx, SP formation, cell viability, and migration and impaired cancer stem-like properties. These findings highlight the critical role of TRPM6/TRPM7-mediated Mg²⁺ transport in CRC progression and emphasize the potential of these channels as therapeutic targets for CRC SP elimination.

INTRODUCTION

Cancer therapy has evolved from surgery, radiation, and chemotherapy to modern molecularly targeted and immunological approaches. Although advances such as targeted inhibitors, antibody drug conjugates, checkpoint blockade, and Chimeric antigen receptor T cells have improved clinical outcomes, challenges including resistance, recurrence, toxicity, and limited accessibility remain significant[1]. Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and second leading cause of cancer-related mortality in men and women worldwide[2]. Despite significant progress in standard therapies, particularly in surgery, chemotherapy, radiotherapy, and molecularly targeted agents, therapeutic resistance, disease recurrence, and metastasis have remained formidable obstacles to curative treatment[1,2]. These limitations have been attributed to the presence of cancer stem cells (CSCs), a subpopulation of tumor-initiating cells that possess self-renewal capabilities, differentiation plasticity, and conventional therapy resistance[3]. As such, therapeutic strategies that target CSCs have become the central focus in CRC research aiming to enhance long-term treatment efficacy and prevent relapse[4].

Recent evidence has implicated dysregulated magnesium (Mg²⁺) homeostasis in the pathogenesis of CRC. Epidemiological studies have demonstrated that low dietary Mg²⁺ intake is associated with increased CRC risk[5-7]. In clinical settings, hypomagnesemia has been frequently observed in patients with CRC, often secondary to targeted therapies such as cetuximab and other anti-epidermal growth factor receptor agents[8-10]. Moreover, CRC tissues exhibit increased intracellular levels of Mg²⁺, potentially due to enhanced sequestration and dysregulated ion transport[11]. Mechanistically, this imbalance is caused by the upregulation of Mg²⁺ influx channels, particularly transient receptor potential melastatin 6 and 7 (TRPM6 and TRPM7), and the concurrent downregulation of the Mg²⁺ efflux transporter cyclin M4[12-14]. These mechanisms cause higher intracellular Mg²⁺ concentrations within tumor tissues than within adjacent nonneoplastic mucosa[12], indicating that Mg²⁺ overload may induce tumor progression.

TRPM7 is a ubiquitously expressed channel-kinase and regulates cellular Mg²⁺ balance, with intracellular Mg²⁺ and Mg-ATP negatively regulating its activity[15-17]. TRPM6 shares similar regulatory mechanisms, although largely restricted to intestinal and renal epithelia[15,18,19]. Notably, heteromeric TRPM6/7 complexes demonstrate reduced sensitivity to intracellular inhibition, enabling sustained Mg²⁺ entry into epithelial cells[20,21], which may cause persistently increased intracellular Mg²⁺ levels in CRC cells. TRPM7 has been attributed to the progression of various cancers, including glioma; leukemia; and ocular, head and neck, esophageal, breast, lung, gastric, liver, pancreatic, colorectal, bladder, ovarian, cervical, prostate, and kidney cancers[22,23]. However, its inhibition in azoxymethane-induced CRC mice did not suppress tumor development, but instead led to systemic Mg²⁺ deficiency[23]. Considering its ubiquitous expression and crucial role in cellular Mg²⁺ homeostasis, TRPM7 antagonism may cause widespread metabolic disturbances. These findings indicate that TRPM7 is not an appropriate therapeutic target for CRC treatment, particularly because of its systemic physiological functions and the risk of inducing systemic Mg²⁺ deficiency. In contrast, the roles of TRPM6 and heteromeric TRPM6/7 channels in CRC progression and CSCs maintenance remain unclear.

Thus, the present study investigated the role of TRPM6 and TRPM6/7 channel inhibition in CRC using the HT-29 cell line. These cells harbor pathogenic mutations in adenomatous polyposis coli, v-raf murine sarcoma viral oncogene homolog B1, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha, SMAD family member 4, and tumor protein p53 and can generate CSCs-enriched spheroids (SPs) exhibiting hallmark traits of stemness, drug resistance, and high self-renewal capability[24]. Moreover, HT-29 SPs display marked resistance to 5-fluorouracil[25], underscoring their use as a robust in vitro model for evaluating novel strategies targeting CSCs and Mg²⁺ homeostasis in CRC.

MATERIALS AND METHODS

Cell culture

Human colorectal adenocarcinoma HT-29 cells (ATCC HTB-38^™, American Type Culture Collection, Manassas, VA, United States) were cultured in Dulbecco’s modified eagle medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, United States) supplemented with 15% fetal bovine serum, 1% L-glutamine, 1% nonessential amino acids, and 1% penicillin-streptomycin (all from Gibco, Thermo Fisher Scientific, Waltham, MA, United States). Cells were maintained under standard conditions at 37 °C in a humidified atmosphere containing 5% CO₂. Subculturing was performed in 75-cm² tissue culture flasks (Corning Inc., NY, United States) following the American Type Culture Collection guidelines. For pharmacological modulation of Mg²⁺ transport, cells were treated with the following inhibitors: Co(III)hexamine (Co(III)hex; Sigma-Aldrich, St. Louis, MO, United States), a broad-spectrum Mg²⁺ channel blocker[26]; 2-aminoethyl diphenylborinate (2-APB; MedChemExpress, Monmouth Junction, NJ, United States), a TRPM6/7 inhibitor[21]; and Mesendogen (ME; MedChemExpress, Monmouth Junction, NJ, United States), a selective TRPM6 inhibitor[27]. A 0.3% (vol/vol) solution of dimethyl sulfoxide was used as the vehicle for inhibitor preparation and was administered to the control group to match the treatment conditions.

Three-dimensional SP culture

HT-29 cells were cultured under non-adherent, serum-free conditions using a 96-well hanging drop system (Perfecta3D^®, Sigma-Aldrich, St. Louis, MO, United States) to promote SP formation. Each well was seeded with 5 × 10⁴ cells in SP medium composed of DMEM/F-12 supplemented with 1X B27 (Gibco, Thermo Fisher Scientific, Waltham, MA, United States), 20 ng/mL of epidermal growth factor (PeproTech, Cranbury, NJ, United States), and 20 ng/mL of basic fibroblast growth factor (PeproTech, Cranbury, NJ, United States). To minimize evaporation, 5 mL of sterile phosphate buffered saline (PBS) was added to the reservoir chamber. Cells were cultured for up to 14 days at 37 °C in an environment containing 5% CO₂. Under these conditions, cells spontaneously aggregated into compact, uniformly spherical structures. Morphological assessment was conducted using inverted bright-field microscopy (Nikon ECLIPSE Ts2-FL; Nikon Instruments Inc., Melville, NY, United States), and quantitative analysis of SP architecture, including Feret diameter, percent area, percent integrated density, and perimeter, was performed using AnaSP version 3.0[28].

Secondary sphere formation assay

To evaluate self-renewal capacity, primary SPs were enzymatically dissociated into single-cell suspensions using trypsin-EDTA (Gibco, Thermo Fisher Scientific, Waltham, MA, United States) followed by gentle pipetting. Viable cells were quantified and re-seeded in SP medium under identical non-adherent conditions. Efficiency of sphere formation was assessed across three serial passages, with consistent seeding density and culture conditions maintained for each generation[24].

Cell viability assay

Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HT-29 cells were seeded into 96-well plates at 4 × 10⁴ cells/well and incubated with MTT reagent (1 mg/mL; Sigma-Aldrich, St. Louis, MO, United States) for 3 hours at 37 °C. Formazan crystals were then solubilized in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, United States), and absorbance was measured at 540 nm using a multimode plate reader (BioTek Instruments, Winooski, VT, United States).

Mg²⁺ influx measurements

Subconfluent cells cultured in μ-Dishes (Ibidi GmbH, Gräfelfing, Germany) were preconditioned for 3 days in DMEM. On the day of measurement, the cells were washed thrice with Ca²⁺- and Mg²⁺-free Dulbecco’s phosphate-buffered saline (HiMedia Laboratories, Mumbai, India) and incubated for 1 hour in HyClone^™ DMEM lacking Ca²⁺, Mg²⁺, L-glutamine, and sodium pyruvate (Cytiva, Marlborough, MA, United States) but reconstituted with 15% fetal bovine serum, 1% l-glutamine, and 1% non-essential amino acids. The cells were then loaded with 5 μmol/L Mag-Fura-2-AM (Invitrogen, Thermo Fisher Scientific, Waltham, MA, United States) in N-methyl-D-glucamine buffer (145 mmol/L N-methyl-D-glucamine, 4.7 mmol/L KCl, 12 mmol/L D-glucose, 2.5 mmol/L l-glutamine, 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; pH 7.4, 290-295 mOsm/kg H₂O) for 1 hour, followed by incubation for 30 minutes in dye-free buffer to allow for complete de-esterification of acetoxymethyl esters[29]. Real-time fluorescence imaging was performed using a confocal laser scanning microscope (FV3000, Olympus Corporation, Tokyo, Japan). Mg²⁺ influx was calculated using the following equation: ΔF/F = [F(t) - F(0)]/[F(0) - F(b)], where F(t) is the mean fluorescence intensity at a given time, F(0) is the basal fluorescence intensity, and F(b) is the background fluorescence[26]. Image analysis was conducted using ImageJ[30].

Quantification of free cellular Mg²⁺

Subconfluent cells cultured in 6-well plates (Corning Inc., NY, United States) were preconditioned for 3 days in DMEM. After being washed three times with sterile PBS, the cells were incubated with a bathing solution (BS) containing (in mmol/L) 118 NaCl, 4.7 KCl, 1.1 MgCl₂, 1.25 CaCl₂, 23 NaHCO₃, 12 D-glucose, 2.5 l-glutamine, and 2 D-mannitol (osmolality: 290-295 mmol/kg H₂O; pH 7.4)[31]. Thereafter, the cells were loaded with 5 μmol/L Mag-Fura-2-AM (Invitrogen, Thermo Fisher Scientific, Waltham, MA, United States) in BS for 30 minutes at 37 °C, washed three times, and then maintained in BS for an additional 30 minutes. Fluorescence signals were acquired using a confocal laser scanning microscope (FV3000, Olympus Corporation, Tokyo, Japan).

Quantification of total cellular Mg²⁺

Total intracellular Mg²⁺ levels were quantified using atomic absorption spectrophotometry (Shimadzu Corporation, Kyoto, Japan), following protocols previously described by Thongon[31]. Briefly, the cells were lysed by sonication, after which the resulting lysates were analyzed against a series of Mg²⁺ standards to determine Mg²⁺ concentrations.

Immunoprecipitation

Immunoprecipitation of TRPM6 (IP-TRPM6) was conducted as described previously[18]. Briefly, the cells were lysed in cold ratchet-integrated pneumatic actuator buffer (Thermo Fisher Scientific, Waltham, MA, United States) containing 10% protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, United States). After centrifugation, membrane proteins were isolated using the Mem-PER^™ Plus Extraction Kit (Thermo Fisher Scientific, Waltham, MA, United States). TRPM6 was immunoprecipitated using a polyclonal anti-TRPM6 antibody (Thermo Fisher Scientific, Waltham, MA, United States) and Protein A/G Sepharose^® beads (Abcam, Cambridgeshire, United Kingdom). Thereafter, protein complexes were incubated overnight at 4 °C, washed, and eluted with glycine-Tris buffer (Sigma-Aldrich, St. Louis, MO, United States). The eluted samples were then concentrated using Vivaspin^® 20 filters (Sartorius AG, Göttingen, Germany) for downstream applications.

Western blotting

Western blot analysis was performed as previously described[18]. Briefly, protein samples were denatured in sodium dodecyl sulfate buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membranes. Precision Plus Protein^™ Dual Xtra Prestained Protein Standards (Cat. No. 1610377; Bio-Rad Laboratories, Hercules, CA, United States) were used as molecular weight markers. The membranes were probed with primary antibodies targeting TRPM6 (PA5-77326; Thermo Fisher Scientific, Waltham, MA, United States), TRPM7 (ab729; Abcam, Cambridgeshire, United Kingdom), matrix metalloproteinase-9 (MMP-9: Ab228402; Abcam, Cambridgeshire, United Kingdom), cyclooxygenase-2 (COX-2: Ab179800; Abcam, Cambridgeshire, United Kingdom), Nanog (ab317506; Abcam, Cambridgeshire, United Kingdom), Na⁺/K⁺-ATPase (ab76020; Abcam, Cambridgeshire, United Kingdom), and β-actin (ab8226; Abcam, Cambridgeshire, United Kingdom), followed by horseradish peroxidase-conjugated secondary antibodies. The SuperSignal^® West Pico chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, United States) was used for detection, whereas a ChemiDoc^™ Touch imaging system (Bio-Rad Laboratories, Hercules, CA, United States) was used for visualization. Densitometric analysis was performed using ImageJ.

Nano-liquid chromatography tandem mass spectrometry analysis

For proteomic profiling, 20 µg of immunoprecipitated TRPM6 was reduced (100 mmol/L DTT), alkylated (100 mmol/L iodoacetamide), and digested with trypsin (Promega Corporation, Madison, WI, United States). Thereafter, the peptides were dried, resuspended in 0.1% formic acid, and subjected to nano-liquid chromatography (LC) tandem mass spectrometry (MS/MS) using a Dionex Ultimate 3000 RSLCnano system (Thermo Fisher Scientific, Waltham, MA, United States) coupled to a Bruker Q-ToF Compact II mass spectrometer (Bruker Daltonics, Billerica, MA, United States). Peptide separation was performed on a PepMap100 C18 column (Thermo Fisher Scientific, Waltham, MA, United States) with a 90-minute acetonitrile gradient at 300 nL/minute. Spectra were then acquired in the positive ion mode and analyzed using the MASCOT engine (version 2.3, Matrix Science, London, United Kingdom) against the UniProtKB database. Quantification was based on the exponentially modified protein abundance index, and post-translational modifications were verified by MS/MS fragmentation.

Wound-healing assay

HT-29 cells were seeded into 24-well plates at 5 × 10⁵ cells/mL and grown to confluence. Afterward, a scratch wound was generated using a sterile cell scraper (Corning Inc., NY, United States), and debris was removed using sterile PBS. Wound closure was monitored over time using a bright-field microscope (Nikon ECLIPSE Ts2-FL; Nikon Instruments Inc, Melville, NY, United States), and the wound area was quantified using ImageJ software.

Statistical analysis

All data were presented as mean ± SD. Comparisons between the two groups were conducted using unpaired Student’s t-test, whereas comparisons between multiple groups were performed via one-way analysis of variance followed by Dunnett’s post-hoc test. Statistical significance was set at P < 0.05. GraphPad Prism (GraphPad Software Inc., San Diego, CA, United States) was used for all analyses.

RESULTS

TRPM6 and TRPM7 expression in cancer SPs

Parental HT-29 monolayers and SP-derived HT-29 cells at comparable culture passages were used. The protein expression levels of TRPM6 and TRPM7 in the total, cytosolic, and membrane fractions are presented in Figure 1A-D. β-actin was utilized as the internal loading control for the total and cytosolic fractions, whereas Na⁺/K⁺-ATPase was the loading control for the membrane protein fractions. Compared to the corresponding fractions in parental HT-29 monolayers, membranous TRPM6 expression was significantly upregulated in SP cells (Figure 1A and E). Similarly, total and membrane TRPM7 were markedly higher in SP cells than in parental cells (Figure 1B and F).

Figure 1 Expression and subcellular localization of transient receptor potential melastatin 6 and transient receptor potential melastatin 7 in parental and spheroid-derived HT-29 cells. A: The western blot analysis of transient receptor potential melastatin (TRPM) 6; B: The western blot analysis of TRPM7; C: TRPM6 in immunoprecipitation of TRPM6 (IP-TRPM6); D: TRPM7 in IP-TRPM6 of parental HT-29 monolayers and spheroid-derived cells; E: The densitometric analysis of TRPM6; F: The densitometric analysis of TRPM7; G: TRPM6 in IP-TRPM6; H: TRPM7 in IP-TRPM6 of parental HT-29 monolayers and spheroid-derived cells. Total: Total cell; Cyto: Cytosol; Mem: Membrane; SP: Spheroid; TRPM: Transient receptor potential melastatin; IP-TRPM6: Immunoprecipitation of transient receptor potential melastatin 6. ^aP < 0.05, ^bP < 0.01, ^cP < 0.01 vs the corresponding parental cells (n = 6).

The membrane fractions were subjected to immunoprecipitation with a TRPM6 antibody (IP-TRPM6), followed by western blot analysis, to evaluate the expression of membrane-bound TRPM6/7 heterodimers. Figure 1C and G show that the parental and SP cells had comparable TRPM6 expression in the IP-TRPM6 membrane fractions, confirming equal protein loading. Subsequent reprobing of the same blots with a TRPM7 antibody (Figure 1D) revealed a significantly stronger TRPM7 signal in SP cells than in parental cells (Figure 1H). These findings indicate that membrane-localized TRPM6, TRPM7, and TRPM6/7 heteromeric channels are upregulated in CRC SPs.

Nano-LC-MS/MS analysis of membranous TRPM6 and TRPM7

Membrane-bound TRPM6 and TRPM7 from membranous IP-TRPM6 protein samples were further analyzed using nano-LC-MS/MS. TRPM7 protein was isolated by decreasing the TRPM6/7 heterodimer in the IP-TRPM6 protein samples. The presence of TRPM6 and TRPM7 in these samples was confirmed by protein sequence analysis. Accordingly, the TRPM6 protein sequence from parental and SP cells were 100% identical to human TRPM6 (UniProtKB: Q9BX84), and the TRPM7 protein sequence from the same groups were 100% identical to human TRPM7 (UniProtKB: Q96QT4). The exponentially modified protein abundance index values for IP-TRPM6 in the parental group were 0.10 for TRPM6 and 0.11 for TRPM7, whereas those in the SP group were 0.96 and 0.10, respectively. These indicate that sufficient and comparable amounts of TRPM6 and TRPM7 proteins were subjected to nano-LC-MS/MS analysis.

Furthermore, MS/MS fragmentation analysis was used to identify phosphorylated residues in full-length TRPM6 and TRPM7 protein sequences. A previous study reported that the S141 residue of TRPM6 regulates the localization and function of the membrane-bound TRPM6/7 heterodimer[32]. S141 residue phosphorylation in TRPM6 was detected in parental and SP cells (Table 1). TRPM6 channel permeability is induced by S1252 residue phosphorylation[33], which was observed only in SP groups. After determining the total number of phosphorylated residues in membranous TRPM6, we found that the parental and SP groups had 158 and 185 phosphorylated residues, respectively. Table 2 lists the phosphorylated residues in membranous TRPM7: 190 in the parental group and 205 in the SP group. Membrane localization and TRPM7 stabilization are modulated by S138 and S1360, respectively[33]. Phospho-S138 and phospho-S1360 residues were detected in membranous TRPM7 in both groups. Additionally, phosphorylated residues modified by the α-kinase domain of TRPM6 were identified (Table 2), the levels of which were comparable in the parental and SP groups. These results demonstrate the hyperphosphorylation of membranous TRPM6 and TRPM7 in HT-29 cancer SPs.

Table 1 Phosphorylated residue of transient receptor potential melastatin 6 proteins.

	Parental HT-29	Spheroid-derived HT-29
N-terminus	S12, S67, S78, Y80, T87, T88, S90, T92, T94, S141, T169, T170, T176, T181, Y228, S243, S246, S274, S282, S302, T380, S386, T398, S407, Y431, S445, Y462, Y479, T481, T509, T530, S552, S558, Y574, S586, S596, T602, Y608, T635, S662, Y697, S705, T706, S743, T756, S820, Y835, Y845, T859, S869	S12, S32, S78, Y80, T87, T88, T94, Y111, Y116, S141, S153, T169, T170, T176, T181, S184, T204, Y228, T230, S236, T239, Y259, S274, S282, T354, S362, T380, S407, S409, Y431, S445, T471, Y506, T509, Y519, Y525, Y529, T530, Y545, S551, S552, S558, Y574, S586, S590, T602, Y645, S655, Y668, S669, S705, T706, S713, S743, T759, S769, S774, S784, S789, S790, S791, S794, S796, S820, Y845, S869 (65)
Channels	T881, S893, T899, T936, S951, T974, S1000, S1006, Y1022, S1038, T1046, Y1053	S876, T881, S893, S907, T913, T915, Y966, T968, T974, Y979, S1008, Y1026, Y1053
TRP	Y1073, S1078, Y1086, Y1089, T1094, Y1095, S1109	Y1073, S1078, S1080, Y1086, Y1089, Y1091, T1094, Y1095
C-terminus	Y1137, S1139, Y1157, S1168, T1231, S1281, S1285, T1322, S1324, S1329, S1349, S1357, T1375, T1391, S1395, S1399, S1736, S1737, T1739, Y1741, S1746, S1747, S1986, T1993, T2011	Y1137, S1139, Y1157, S1168, T1231, S1244, T1245, S1252, S1254, Y1273, S1277, S1281, S1285, T1311, S1332, S1339, Y1341, S1368, T1375, S1736, S1737, T1739, Y1741, S1746, S1747, S1970, Y1984, T1992, S2002, T2011, S2015
Coiled coil	T1176, S1177, T1181, Y1184, S1195, S1200, S1206, T1221, S1226	T1176, S1177, T1181, Y1184, S1195, S1200, S1206, T1221, S1226
S/T rich domain	S1438, S1441, S1458, T1463, S1487, S1498, S1500, S1503, Y1533, S1539, S1541, T1577, T1598, S1603, S1616, S1618, S1623, T1628, S1630, S1633, T1635, T1647, Y1660, S1672, T1679, S1685, S1689, S1690, S1697, S1699, S1702, T1739, Y1741	T1435, S1437, S1467, T1474, S1478, T1479, S1498, S1500, T1523, Y1533, S1539, S1541, S1562, S1563, T1577, S1583, T1589, S1605, Y1622, S1623, T1628, S1630, S1658, S1664, S1669, S1678, T1679, S1685, S1689, S1690, S1697, S1699, S1702, T1739, Y1741
Dimerization motif	Y1710, S1711, S1722, T1724, T1728, Y1731	Y1710, S1711, S1722, T1724, T1728, Y1731
α-kinase domain	S1754, S1757, S1821, T1822, T1843, Y1854, Y1878, Y1880, T1895, T1897, S1908, Y1912, Y1914, T1915, T1932, S1935	S1754, S1759, S1771, S1787, S1790, T1813, T1822, T1843, Y1854, T1855, Y1865, S1868, Y1886, S1908, Y1912, Y1914, T1915, S1944

TRP: Transient receptor potential.

Table 2 Phosphorylated residue of transient receptor potential melastatin 7 proteins.

	Parental HT-29	Spheroid-derived HT-29
N-terminus	S9, T10, T12, Y18, S22, S23, T55, S57, Y62, S79, T84, S87, T89, S101, S103, Y104, Y108, S113, T115, S138, S193, S196, S233, S243, Y256, T270, Y303, T318, Y327, T332, T349, T353, S359, T379, S385, T397, S406, Y430, S453, S464, T470, Y505, T508, Y528, S539, S547, S552, S554, S561, T564, T583, Y587, T603, T615, S648, S683, T737, T739, S745, T778, S788, T807 S823, S824, S836, Y849	S2, S9, T10, Y18, S22, S23, T55, S57, Y62, S63, S74, S79, S87, T89, Y92, S101, S103, Y108, S112, Y113, T115, S138, T166, T167, T173, S195, S196, T201, Y225, T227, S243, T252, T269, T318, T332, T353, S359, T367, T379, Y430, S438, S453, S464, T508, S539, S547, S552, S553, S554 T555, S561, T564, T583, Y587, T603, T615, S648, S661, S676, S727, S728, T778, S783, S788, T795, S799, S823, S824, S836, Y849, T860, Y863
Channels	T895, Y896, S913 S921, S927, T929, S934, Y965, Y989, Y1023, S1031, Y1051, S1060, T1064, T1070, T1073, Y1080, Y1085	T895, Y896, S913, S921, S927, S929, Y949, Y953, Y989, Y1002, S1013, Y1023, S1029, S1031, Y1041, Y1051, S1060, T1064, Y1080, Y1085
TRP	Y1100, S1107, Y016, Y1122, S1036, S1040, S1141	Y1100, S1107, Y016, Y1122, S1036, S1040, S1141
C-terminus	T1154, S1155, T1163, Y1181, S1191, S12551, S1258, T1265, S1269, S1271, S1292, T1296, S1298, S12991, S13001, S1308, S13491, S1350, S13601, S1587, S1888, S1590, Y1826, T18271, S1839, S1848, T1849, T1855	T1154, S1155, T1163, Y1181, S1191, S1193, S12551, S1258, T1265, S1269, S1271, S1292, T1296, S1298, S12991, S13001, S1308, S13491, S1350, S1357, S13601, T1580, S1587, S1588, S1590, Y1826, T18271, S1839, S1848, T1849, S1852, T1855, S1857
Coiled coil	T1200, S1208, S12241, S1227, T1242, T1245, T1248, T12501	T1200, Y1220, S12241, S1227, S1230, S1239, T1242, T12501
S/T rich domain	S13861, S1389, S13901, S1394, S13951, S14031, T1404, S1406, S1409, S1412, S14161, T1418, S14451, S1455, S1463, T14661, T1470, Y1479, T1485, T14871, S14881, S14911, T1493, S14951, S14971, S15011, T15021, S15051, S15101, T1524, S1530, T1534, S15401, T1548	S13861, S1389, S13901, S1394, S13951, S14031, T1404, S1406, S1409, S1412, S14161, T1418, Y1426, S14451, T1454, S1455, S1463, T14661, T1470, Y1479, T1485, T14871, S14881, S14911, T1493, S14951, S14971, S15011, T15021, S15051, S15101, T1524, S1530, T1534, S15401, T1548
Dimerization motif	S1553, S15641, S15661	S1553, S15641, S15661
α-kinase domain	S1595, S1597, S1598, S1600, S1612, T1629, S1631, Y1642, S1656, S1657, Y1659, T1663, S16921, Y1695, Y1696, S1709, Y1727, T1738, T1740, S1749, T1756, S1776, S1785	S1592, S1597, S1598, S1600, S1612, T1629, S1638, S1646, S1656, Y1659, T1663, T1682, S16921, Y1695, Y1696, S1709, Y1727, T1738, T1740, S1749, T1752, Y1753, Y1755, T1756, S1776, S1782, S1811

¹Phosphorylated residues modified by the α-kinase domain of transient receptor potential melastatin 6.

TRP: Transient receptor potential.

MS/MS fragmentation analysis further revealed widespread methionine oxidation in the full-length sequences of membrane-localized TRPM6 and TRPM7 proteins (Table 3). Notably, TRPM6 in SP and parental cells exhibited oxidation at 33 and 30 methionine residues, respectively. Similarly, TRPM7 in SP and parental cells displayed oxidation at 30 and 28 methionine residues, respectively. These findings show a modest but consistent increase in oxidative modification of TRPM channels in SP cells, potentially reflecting increased oxidative stress levels commonly associated with CRC progression[34].

Table 3 Methionine oxidation in transient receptor potential melastatin 6 and transient receptor potential melastatin 7 proteins.

TRPM6		TRPM7
Parental HT-29	Spheroid-derived HT-29	Parental HT-29	Spheroid-derived HT-29
M33, M63, M133, M338, M370, M416, M618, M623, M625, M648, M657, M732, M768, M847, M864, M969, M984, M1020, 1061, M1076, M1093, M1162, M1278, M1183, M1434, M1436, M1575, M1879, M2020	M1, M33, M133, M244, M338, M370, M416, M444, M450, M618, M623, M625, M648, M657, M727, M734, M739, M768, M847, M969, M984, M1093, M1162, M1190, M1265, M1434, M1436, M1575, M1719, M1879, M1904, M1947, M2020	M43, M372, M632, M637, M649, M706, M742, M746, M782, M794, M812, M868, M878, M906 M991, M996, M1000, M1088, M1020, M1180, M1207, M1287, M1319, M1373, M1446, M1616, M1720, M1745	M130, M575, M591, M595, M649, M662, M704, M706, M741, M746, M773, M782, M812, M868, M991, M992, M996, M1000, M1180, M1207, M1287, M1318, M1373, M1446, M1528, M1561, M1616, M1720, M1745, M1788

TRPM: Transient receptor potential melastatin.

Mg²⁺ influx and total Mg²⁺ content in the cancer SPs

The ΔF/F fluorescence signal was measured during the first 30 seconds of the preincubation period and from 40 to 160 seconds after adding 20 mmol/L of MgSO₄. Upon MgSO₄ addition, the intracellular fluorescence signal in the parental HT-29 cells rapidly increased, followed by a plateau phase (Figure 2). Mg²⁺ influx was significantly greater in SP cells than in parental cells, consistent with the upregulation of membranous TRPM6 and TRPM7 expression. Kinetic analysis of the ΔF/F signal (40-160 seconds) using the Michaelis-Menten equation (Figure 2) revealed that the maximum reaction rate (Vmax) was significantly higher in SP cells (1.81 ± 0.09) than in parental cells (0.78 ± 0.06; P < 0.001). Furthermore, the total intracellular Mg²⁺ levels were significantly higher in SP cells than in parental HT-29 cells (Figure 2). However, the free ionized intracellular Mg²⁺ levels remained comparable across all experimental groups (Figure 2).

Figure 2 Mg²⁺ influx and total Mg content in cancer spheroids. A: Mg²⁺ influx in parental HT-29 cells compared with spheroid (SP)-derived HT-29 cells; B: Mg²⁺ influx in SP cells with or without the Mg²⁺ channel inhibitor Co(III)hexamine; C: Mg²⁺ influx in SP cells with or without the transient receptor potential melastatin 6/7 inhibitor 2-aminoethyl diphenylborinate; D: Mg²⁺ influx in SP cells with or without the transient receptor potential melastatin 6 inhibitor Mesendogen; E: Michaelis-Menten kinetic analysis of ΔF/F values between 40 and 160 seconds in parental HT-29 vs SP cells, SP vs SP with Co(III)hexamine, SP vs SP with 2-aminoethyl diphenylborinate, and SP vs SP with Mesendogen; F: Total intracellular Mg²⁺ content; G: Free intracellular Mg²⁺ level. SP: Spheroid; Co(III)hex: Co(III)hexamine; 2-APB: 2-aminoethyl diphenylborinate; ME: Mesendogen. ^aP < 0.05 vs parental HT-29 cells, ^bP < 0.01 vs parental HT-29 cells, ^cP < 0.001 vs parental HT-29 cells, ^dP < 0.05 vs spheroid group, ^eP < 0.01 vs spheroid group, and ^fP < 0.001 vs spheroid group.

Subeffective, effective, and supra-effective concentrations of inhibitors were employed to investigate the involvement of TRPM6 and TRPM6/7 channels in enhancing Mg²⁺ influx and total Mg²⁺ content in SP cells. To this end, the effects of Co(III)hex (10 μmol/L, 100 μmol/L, and 1 mmol/L), 2-APB (10 μmol/L, 200 μmol/L, and 1 mmol/L), and ME (0.5 μmol/L, 10 μmol/L, and 500 μmol/L) on parental and SP cell viability were evaluated. After both cell types were treated for 24 hours, MTT assay analysis was performed. Co(III)hex (Figure 3A), 2-APB (Figure 3B), and ME (Figure 3C) treatment had no effect on parental HT-29 cell viability at any of the tested concentrations. In contrast, treatment with these inhibitors significantly decreased SP cell viability.

Figure 3 Effects of transient receptor potential melastatin inhibitors on the viability of spheroid-derived cells. A: Cell viability of parental and spheroid (SP)-derived HT-29 cells treated with the Mg²⁺ channel inhibitor Co(III)hexamine; B: Cell viability of parental and SP cells treated with the transient receptor potential melastatin 6/7 inhibitor 2-aminoethyl diphenylborinate; C: Cell viability of parental and SP cells treated with the transient receptor potential melastatin 6 inhibitor Mesendogen; D: Cell viability of SP cells with or without Co(III)hexamine, 2-aminoethyl diphenylborinate, or Mesendogen treatment for 12 hours. SP: Spheroid; Co(III)hex: Co(III)hexamine; 2-APB: 2-aminoethyl diphenylborinate; ME: Mesendogen. ^aP < 0.05, ^bP < 0.01 vs the control spheroid group (n = 6).

The effects of a 12-hour incubation with effective doses of Co(III)hex (100 μmol/L), 2-APB (200 μmol/L), and ME (10 μmol/L) on SP cell viability were further assessed. However, no significant decrease in SP cell viability was observed following the 12-hour treatments (Figure 3D). Mg²⁺ influx in SP cells was examined after the 12-hour incubation with the mentioned inhibitors. The cells treated with Co(III)hex (Figure 2), 2-APB (Figure 2), and ME (Figure 2) showed significantly suppressed Mg²⁺ influx compared to control SP cells. The Vmax values for the groups treated with Co(III)hex (0.64 ± 0.07; P < 0.001), 2-APB (0.62 ± 0.06; P < 0.001), and ME (0.99 ± 0.10; P < 0.01) were markedly lower than those for control SP cells (1.81 ± 0.09; Figure 2). Moreover, 12 hours treatment with Co(III)hex, 2-APB, and ME significantly decreased the total intracellular Mg²⁺ levels in SP cells (Figure 2). These findings indicate that Co(III)hex, 2-APB, and ME inhibit Mg²⁺ influx, decrease intracellular Mg²⁺ accumulation, and decrease the viability of CRC SPs.

TRPM6 and TRPM6/7 inhibitor disrupt cancer SP formation

Figure 4 shows that well-defined CRC SPs were successfully generated using the hanging drop method within 14 days of seeding. HT-29 cells aggregated into compact, round SPs that progressively matured over time, with denser central core and clearly demarcated edges forming by 24 and 48 hours of culture, respectively. This morphological integrity indicates stable cancer SP formation under standard conditions. In contrast, treatment with Mg²⁺ channel inhibitors Co(III)hex, 2-APB, and ME markedly disrupted SP integrity. Within 12 hours of exposure to effective concentrations, the peripheral cells began to detach from the SP structure, indicating early destabilization of intercellular cohesion. After 24 hours of treatment with effective and supra-effective concentrations of Co(III)hex (Figure 4A-E), 2-APB (Figure 4F-J), or ME (Figure 4K-O), the SPs exhibited complete structural collapse, with disaggregation of the cells and loss of their spherical architecture. These demonstrate that pharmacological inhibition of TRPM6- and TRPM6/7-mediated Mg²⁺ influx significantly impairs the formation and maintenance of CRC SPs, emphasizing the importance of Mg²⁺ homeostasis regulated by TRPM channels for sustaining the structural integrity and viability of cancer SPs.

Figure 4 Transient receptor potential melastatin inhibitors affect spheroid stability. A-E: Cancer spheroids treated with or without Co(III)hexamine, representative images, percent integrated density, percent spheroid area, Feret diameter, perimeter; F-J: Cancer spheroids treated with or without 2-aminoethyl diphenylborinate, representative images, percent integrated density, percent spheroid area, Feret diameter, perimeter; K-O: Cancer spheroids treated with or without Mesendogen, representative images, percent integrated density, percent spheroid area, Feret diameter, perimeter. Scale bar = 200 μm. SP: Spheroid; Co(III)hex: Co(III)hexamine; 2-APB: 2-aminoethyl diphenylborinate; ME: Mesendogen. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs the same grout at time 0 hour (n = 6).

A secondary sphere formation assay was performed to evaluate the self-renewal capacity of CSCs-enriched HT-29 cancer SPs. It was found that SP cells retained the ability to form secondary SPs even after three successive subcultures (Figure 5A), confirming the CSCs-enriched characteristics of HT-29 CRC cells. Conversely, treatment with 100 μmol/L Co(III)hex, 200 μmol/L 2-APB, or 10 μmol/L ME markedly suppressed secondary SP formation over a 14-day period using the hanging drop culture system. Moreover, after assessing the expression of the CSC marker Nanog[35] (Figure 5B and C), all three inhibitors were found to significantly decrease Nanog expression in SP cells. These findings indicate that inhibiting Mg²⁺ influx and reducing total cellular Mg²⁺ levels may eliminate the CSCs-like properties of HT-29 CRC cells.

Figure 5 Secondary sphere formation assay. A: Serial spheroid formation was assessed by repeated passaging of cancer spheroid (SP)-derived cells treated with or without 100 μmol/L Co(III)hexamine, 200 μmol/L 2-aminoethyl diphenylborinate, or 10 μmol/L Mesendogen. Scale bar = 200 μm; B: The western blot analysis; C: Densitometric analysis of Nanog in SP cells treated with or without 100 μmol/L Co(III)hexamine, 200 μmol/L 2-aminoethyl diphenylborinate, or 10 μmol/L Mesendogen. SP: Spheroid; Co(III)hex: Co(III)hexamine; 2-APB: 2-aminoethyl diphenylborinate; ME: Mesendogen. ^aP < 0.05, ^bP < 0.01 vs the control spheroid group (n = 6).

TRPM6 and TRPM6/7 inhibitors suppressed cancer cell migration

The migratory capacity of SP HT-29 cells was investigated using a two-dimensional wound-healing assay. After cell treatment using Mg²⁺ channel inhibitors Co(III)hex, 2-APB, or ME and monitoring for wound closure over a 6-24 hours period, all three treatment groups exhibited significantly decreased wound closure compared to the control group, as evidenced by larger remaining wound areas at each time point (Figure 6). Quantitative analysis confirmed that inhibiting Mg²⁺ influx promoted a sustained delay in cell migration. These findings show that Mg²⁺ homeostasis, which is maintained through TRPM6 and TRPM6/7 channel activity, is crucial for the migratory behavior of CRC cells. Pharmacological disruption of Mg²⁺ influx decreases total intracellular Mg²⁺ levels and impairs the cellular mechanisms required for efficient wound closure, emphasizing a potential therapeutic avenue for suppressing CRC cell migration.

Figure 6 Effects of Mg²⁺ channel inhibitors on migration of spheroid-derived HT-29 cells. A and B: Wounding area of spheroid (SP)-derived HT-29 cells treated with or without Co(III)hexamine; C and D: Wounding area of SP-derived HT-29 cells treated with or without 2-aminoethyl diphenylborinate; E and F: Wounding area of SP-derived HT-29 cells treated with or without Mesendogen. Scale bar = 200 μm. Co(III)hex: Co(III)hexamine; 2-APB: 2-aminoethyl diphenylborinate; ME: Mesendogen. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs the corresponding control group at same time point (n = 6).

Additionally, the expression of COX-2 and MMP-9, which are well-established biomarkers of CRC, was examined[36,37]. Studies have implicated COX-2 in promoting CSCs-like activity and in contributing to apoptotic resistance, proliferation, angiogenesis, inflammation, invasion, and metastasis[38]. Similarly, evidence has shown that MMP-9 facilitates tumor progression and metastatic dissemination[37]. Figure 7 shows that COX-2 and MMP-9 were highly expressed in SP cells, indicating enhanced malignant potential. In contrast, treatment with 100 μmol/L Co(III)hex, 200 μmol/L 2-APB, or 10 μmol/L ME markedly suppressed the expression of both biomarkers. These results indicate that inhibiting Mg²⁺ influx via TRPM6 and TRPM6/7 channels can attenuate CRC aggressiveness by downregulating pro-tumorigenic markers, such as COX-2 and MMP-9.

Figure 7 Effects of Mg²⁺ channel inhibitors on the expression of cyclooxygenase-2 and matrix metalloproteinase-9. A and B: The western blot analysis and densitometric analysis of cyclooxygenase-2 in spheroid cells treated with or without 100 μmol/L Co(III)hexamine, 200 μmol/L 2-aminoethyl diphenylborinate, or 10 μmol/L Mesendogen; C and D: The western blot analysis and densitometric analysis of matrix metalloproteinase-9 in spheroid cells treated with or without 100 μmol/L Co(III)hexamine, 200 μmol/L 2-aminoethyl diphenylborinate, or 10 μmol/L Mesendogen. SP: Spheroid; Co(III)hex: Co(III)hexamine; 2-APB: 2-aminoethyl diphenylborinate; ME: Mesendogen; COX-2: Cyclooxygenase-2; MMP-9: Matrix metalloproteinase-9. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs the control spheroid group (n = 6).

DISCUSSION

The current study emphasizes the critical roles of membrane-localized TRPM6 and TRPM6/7 channels in CRC SP formation, cancer stemness, and cell migration. Our data revealed marked upregulation of membranous TRPM6, TRPM7, and heterodimer TRPM6/7 expression in SP cells, which corresponded with significantly enhanced Mg²⁺ influx. Moreover, kinetic analysis using the Michaelis-Menten model showed a higher Vmax in SP cells than in parental HT-29 cells, indicating an increased number of functional TRPM channels in the plasma membrane. Considering that Vmax is directly related to the number of active transporters, this finding confirms the upregulation and membrane localization of TRPM6, TRPM7, and TRPM6/7 in SP cells.

Total intracellular Mg²⁺ levels were also increased in SP cells, which is consistent with the findings of previous reports linking TRPM6/TRPM7 expression and Mg²⁺ content with CRC progression[12,13]. Although TRPM7 has been shown to regulate CRC cell proliferation and invasion[22], our findings enhance this knowledge by showing that TRPM6 and TRPM6/7 heterodimers are central mediators of SP stability and metastatic behavior. Inhibiting Mg²⁺ influx using Co(III)hex, 2-APB, or ME decreased intracellular Mg²⁺ content and impaired CRC SP formation and cell migration.

The secondary sphere formation assays confirmed that SP cells possess CSCs-like properties, as evidenced by the sustained SP-forming ability across multiple passages. Mg²⁺ channel inhibition disrupted this capacity and downregulated Nanog expression, a key marker of CSCs. These findings support the hypothesis that Mg²⁺ influx through TRPM6 and TRPM6/7 is critical for maintaining CSCs functionality, providing a novel perspective on the role of Mg²⁺ in tumorigenesis[4].

The specific mechanisms through which decreased Mg²⁺ influx and content disrupt SP formation and migration remain unclear. However, studies have well established that cancer cells exhibit high mitochondrial energy demands[39], with Mg²⁺ serving as a crucial cofactor in mitochondrial bioenergetics, particularly in ATP production and oxidative phosphorylation[40]. Thus, decreased intracellular Mg²⁺ upon TRPM6 and TRPM6/7 inhibition may impair mitochondrial function, subsequently compromising cell viability, proliferation, and migration capabilities. Additionally, intracellular Mg²⁺ increases during the G1 and S phases of the cell cycle, potentially facilitating G1/S transition[41], a process frequently dysregulated in cancer[42]. Thus, TRPM6 and TRPM6/7 inhibition may impair metabolic and cell cycle progression, thereby reducing viability and invasive capacity.

Nano-LC-MS/MS analysis provided further insights into posttranslational modifications of membranous TRPM6 and TRPM7 proteins. Phospho-S141 on TRPM6 was detected, which is implicated in regulating TRPM6/7 heterodimerization, in both the parental and SP groups[32]. Phospho-S1252 on TRPM6, which enhances channel permeability[33], was observed specifically in SP HT-29 cells, indicating the increased Mg²⁺ influx observed in this group. Phospho-S138 and phospho-S1360 residues on TRPM7, known to stabilize plasma membrane localization[32,33], were identified in the parental and SP cells. Interestingly, the SP cells demonstrated hyperphosphorylation at these sites, revealing that posttranslational modifications may further enhance channel activity or membrane retention in CRC SPs. However, the functional consequences of this hyperphosphorylation on channel activity or tumorigenic behavior remain to be determined. Studies have identified oxidative stress as a hallmark of CRC pathogenesis[34]. Accordingly, our findings demonstrated methionine hyperoxidation in TRPM6 and TRPM7 proteins of SP cells. Although the functional impact of methionine oxidation on the TRPM channel activity remains unknown, oxidative modifications have generally been associated with altered protein function, including impaired ion conductance and protein stability. These aspects warrant further investigation.

Although Mg²⁺ homeostasis dysregulation is well recognized in cancer[11], the relationship between Mg²⁺ status and colorectal carcinogenesis remains complex and controversial. A previous meta-analysis reported that insufficient and excessive dietary Mg²⁺ intake were associated with increased CRC risk, indicating a biphasic or U-shaped relationship between Mg²⁺ intake and tumorigenesis[43]. In contrast, a recent study using CRC mouse models showed that Mg²⁺ supplementation significantly suppressed tumor growth[44], highlighting the influence of dietary Mg²⁺ intake and extracellular Mg²⁺ levels on CRC progression. However, the present study reveals that increased total intracellular Mg²⁺ levels may promote CRC progression, particularly by enhancing SP stability and migratory potential. Pharmacological inhibition of the TRPM6 and TRPM6/7 channels, which mediate colonocyte Mg²⁺ influx, effectively decreased intracellular Mg²⁺ content and suppressed cancer SP formation and cell migration. Similarly, Castiglioni et al[45] reported that doxorubicin-resistant CRC LoVo cells displayed significantly higher total intracellular Mg²⁺ levels than their sensitive counterparts, further indicating a potential link between intracellular Mg²⁺ accumulation and treatment resistance. Thus, further studies are required to clearly delineate the distinct roles of systemic (dietary or serum) and intracellular Mg²⁺ levels in CRC initiation, progression, and treatment response.

To date, direct evidence demonstrating that TRPM6 and TRPM6/7 channel inhibitors regulate pro-tumorigenic markers, such as Nanog, COX-2, and MMP-9 in CRC, is limited. However, enhanced Mg²⁺ influx through TRPM6 and TRPM6/7 channels potentially increases total intracellular Mg²⁺ levels, which may consequently activate intracellular signaling pathways, such as phosphoinositide 3-kinase (PI3K)[46,47]. Notably, the PI3K pathway has been implicated in the transcriptional regulation of various pro-oncogenic markers, including Nanog[48], COX-2[49], and MMP-9[50]. These proteins are involved in cancer stemness, inflammation, invasion, and metastasis. Although our findings indicate that TRPM6 and TRPM6/7 inhibition downregulates these markers, our hypothesized underlying molecular mechanisms, particularly Mg²⁺-dependent PI3K signaling involvement, require in-depth further investigation to establish a causal relationship.

The role of TRPM7 in cancer diagnosis, development, progression, invasion, treatment response, and prognosis has been comprehensively reviewed by Liu et al[22]. Owing to its ubiquitous expression and essential function in regulating intracellular Mg²⁺ homeostasis[15-17], systemic TRPM7 antagonism may broadly disrupt Mg²⁺ balance across multiple organ systems. As Mg²⁺ plays a critical role in various biochemical and cellular processes, its deficiency has been implicated in some pathological conditions[51]. Although TRPM7 antagonists have demonstrated potential in suppressing the progression of various cancers[22], their off-target effects on systemic Mg²⁺ regulation may result in significant adverse effects. For example, TRPM7 inhibition in azoxymethane-induced CRC mouse models failed to suppress tumor development, but instead led to hypomagnesemia and bone Mg²⁺ depletion, indicating systemic Mg²⁺ deficiency[23]. Approximately 50%-60% of total body Mg²⁺ is stored in bone; Mg²⁺ deficiency can contribute to osteoporosis by impairing bone formation and enhancing bone resorption[51]. Therefore, a more selective therapeutic strategy that targets cancer-specific TRPM7 phenotypes or tumor-restricted TRPM7 signaling pathways is crucial to maximize anticancer efficacy while minimizing systemic toxicity. Conversely, TRPM6 and heteromeric TRPM6/7 channels are exclusively expressed in intestinal and renal epithelia[51]. Specific TRPM6 or TRPM6/7 antagonists formulated with a colon-targeted release coating and low intestinal absorption may suppress CRC progression while minimizing systemic side effects. However, the roles of TRPM6 and TRPM6/7 in CRC progression and their potential systemic adverse effects require further investigation.

Recent perspectives highlight that effective cancer therapy requires a comprehensive strategy that targets not only tumor cells but also the supportive stromal and immune components of the tumor microenvironment[52]. Because direct tumor-focused approaches are often limited by genomic instability and therapeutic resistance, stromal and immune cells have been proposed as more stable targets capable of either promoting or restraining tumor growth. Previous studies also suggest a role for Mg²⁺ in regulating immune and stromal cell functions[53,54]. Nevertheless, the contribution of TRPM-mediated intracellular Mg²⁺ homeostasis to tumor microenvironment dynamics remains to be fully elucidated.

Recent advances in circulating tumor DNA analysis have provided highly sensitive and specific platforms for detecting oncogenic mutations in plasma. Zhang et al[55] demonstrated that programmable endonuclease-mediated cleavage can selectively enrich rare NRAS mutant fragments from a background of wild-type DNA, enabling detection at frequencies below 0.1%. This technological breakthrough underscores the potential of liquid biopsy to expand molecular diagnostics beyond tissue-based sequencing. Within this framework, TRPM6 and TRPM7 emerge as promising candidates for circulating tumor DNA-based biomarker development, given their established roles in Mg²⁺ homeostasis, cell proliferation, and oncogenic signaling in CRC. However, their utility in liquid biopsy applications requires further comprehensive investigation.

Recent literature on traditional medicine highlights the potential of ion channels as novel therapeutic targets in cancer. Toxin-derived compounds, such as scorpion extracts, have been shown to exert antitumor effects through ion channel regulation, underscoring the need to delineate underlying molecular mechanisms to ensure both efficacy and safety[56,57]. Clinical and mechanistic studies have further demonstrated that phytochemicals and complex herbal formulations can modulate tumor growth and treatment response[58,59]. These insights align with our findings on TRPM6 and TRPM6/7, which serve as organ-specific Mg²⁺ transport channels in CRC. Accordingly, traditional medicine-inspired strategies that harness ion channel modulation provide a conceptual framework for advancing TRPM6/7 as precision-oriented therapeutic targets in oncology.

CONCLUSION

TRPM6 and TRPM6/7 channels play a critical role in promoting CRC SP formation and migration. Considering that TRPM6 and TRPM6/7 channels are predominantly expressed in gastrointestinal tissues, their selective inhibition is a promising strategy for CRC treatment, which could potentially minimize systemic side effects. Nonetheless, further preclinical studies are warranted to validate these targets and explore their therapeutic potential.

ACKNOWLEDGEMENTS

We would like to express our deepest gratitude to Seelaphong P of the Microscopic Center, Faculty of Science, Burapha University and Pannengpetch S of the Center for Research and Innovation, Faculty of Medical Technology, Mahidol University for their excellent technical assistance.