Inorganic Polyphosphate Promotes Colorectal Cancer Growth via TRPM8 Receptor Signaling Pathway

Abstract

Background: Colorectal cancer (CRC) is characterized by a pro-inflammatory microenvironment and features high-energy-supply molecules that assure tumor growth. A still less studied macromolecule is inorganic polyphosphate (iPolyP), a high-energy linear polymer that is ubiquitous in all forms of life. Made up of hundreds of repeated orthophosphate units, iPolyP is essential for a wide variety of functions in mammalian cells, including the regulation of proliferative signaling pathways. Some evidence has suggested its involvement in carcinogenesis, although more studies need to be pursued. Moreover, iPolyP regulates several homeostatic processes in animals, spanning from energy metabolism to blood coagulation and tissue regeneration.

Results: In this study, we tested the role of iPolyP on CRC proliferation, using in vitro and ex vivo approaches, in order to evaluate its effect on tumor growth. We found that iPolyP is significantly increased in tumor tissues, derived from affected individuals enrolled in this study, compared to the corresponding peritumoral counterparts. In addition, iPolyP signaling occurs through the TRPM8 receptor, a well-characterized Na⁺ and Ca²⁺ ion channel often overexpressed in CRC and linked with poor prognosis, thus promoting CRC cell proliferation. The pharmacological inhibition of TRPM8 or RNA interference experiments performed in established CRC cell lines, such as Caco-2 and SW620, showed that the involvement of TRPM8 is essential, greater than that of the other two known iPolyP receptors, P2Y1 and RAGE. The presence of iPolyP drives cancer cells towards the mitotic phase of the cell cycle by enhancing the expression of ccnb1, which encodes the Cyclin B protein. In vitro 2D and 3D data reflected the ex vivo results, obtained by the generation of CRC-derived organoids, which increased in size.

Conclusions: These results indicate that iPolyP may be considered a novel and unexpected early biomarker supporting colorectal cancer cell proliferation.

Keywords: TRPM8 receptor; ccnb1; colorectal cancer; inorganic polyphosphate; organoids; proliferation.

Figure 1

Box plot of iPolyP distribution, stratified by peritumoral or tumoral patients. A statistical difference was found (median value of 373,097.70 ± 210,216.20 pmol/mg for tumoral tissue vs. 166,102.70 ± 124,618.80 pmol/mg for the peritumoral counterpart, **** p < 0.0001). Analysis was performed by the Mann–Whitney rank test.

Figure 2

CRC displays a high level of PCNA. (A). Cellular extracts from 50 human biopsies, where the tumoral sample (T) was plotted against the peritumoral (P) counterpart of the same patient (Pt), were analyzed by immunoblotting for the PCNA expression level. Actin was used as a loading control for the normalization. The uncropped bolts are shown in Supplementary Materials. (B). The Spearman correlation between iPolyP and PCNA in the total cohort, denoting a strong positive correlation (**** p < 0.0001). Fold changes versus peritumoral (P), normalized to 1.

Figure 3

CRC displays a high level of TRPM8 receptor. (A). Cellular extracts from 50 human biopsies, where the tumoral sample (T) was plotted against the peritumoral (P) counterpart of the same patient (Pt), were analyzed by immunoblotting for the TRPM8 expression level. Actin was used as a loading control for the normalization. The uncropped bolts are shown in Supplementary Materials. (B). The Spearman correlation between iPolyP and TRPM8 in the total cohort, denoting a strong positive correlation (**** p < 0.0001). Fold changes versus peritumoral (P), normalized to 1.

Figure 4

iPolyP enhances PCNA expression and promotes Caco-2 colorectal cancer proliferation. (A). Cellular extracts from WT and siRNA-mediated TRPM8 knockdown Caco-2 cell lines were analyzed by immunoblotting for the PCNA expression level. GAPDH was used as a loading control. The uncropped bolts are shown in Supplementary Materials. (B). Representative micrographs of the crystal violet assay performed on WT and siRNA-mediated TRPM8 knockdown Caco-2 cell lines upon treatment for 96 h with iPolyP, a TRPM8 inhibitor, or both. Scale bar, 10 µm. Images are representative of three independent experiments. (C). Statistical analysis of the crystal violet assay by Student’s t-test, respectively, for panel (C) (*** p < 0.001 and **** p < 0.0001). Fold changes versus control, untreated (UT), normalized to 1. Data are presented as the mean ± SD for triplicate wells from three independent experiments.

Figure 5

iPolyP promotes colorectal cancer patient-derived organoids and Caco-2- and SW620-derived 3D spheroids. (A,B). Two independent experiments of a CRC patient’s derived organoids, assessed by light microscopy, in the absence of iPolyP (UT) or incubated for 10 days in the presence of iPolyP. Scale bar, 100 µm. Fold changes versus control, untreated (UT), normalized to 1. Statistical analysis was performed by Student’s t-test (*** p < 0.001). (C). Representative bright-field images of 24 h- and 96 h-induced spheroid formation derived from the HCEC-1CT, Caco-2, and SW620 cell line, respectively, upon treatment with iPolyP, a TRPM8 inhibitor, or both for 96 h. Scale bar, 100 µm. Images are representative of three independent experiments. (D). Quantification relative to panel (C). Fold changes versus control, untreated (UT). Statistical analysis was performed by Student’s t-test (**** p < 0.0001). Data are presented as the mean ± SD for triplicate wells from three independent experiments. The uncropped bolts are shown in Supplementary Materials.

Figure 6

iPolyP drives cells into G2/M phase. (A) Real-time PCR on the iPolyP-treated Caco-2 cell line for 72 h on cyclins implicated in different phases of the cell cycle; untreated (UT) samples were normalized to 1. * p < 0.05; ** p < 0.01; **** p < 0.0001. (B) Sketch representing cyclins-dependent cell-cycle regulation consisting of Gap 1 (G1), synthesis (S), Gap 2 (G2), and mitosis (M). Figure was created with BioRender. (C) Representative micrographs of the cell-cycle assay on Caco-2 (upper panel) and SW620 (lower panel) cell line treated for 72 h with iPolyP, a TRPM8 inhibitor, or both. Scale bar = 10 µm. Images are representative of three independent experiments. (D) Percentage of cells in G2/M phase. Statistical analysis was performed by Student’s t-test (*** p < 0.001). Fold changes versus control, untreated (UT). Data are presented as the mean ± SD for triplicate wells from three independent experiments.

Figure 7

Schematic of the role of iPolyP derived from platelets and microbiota on macrophages and CRC cancer cells. The iPolyP–TRPM8 axis stimulates colorectal cancer expansion by enhancing the level of the ccnb1 gene, which coordinates the M phase of the cell cycle, alongside the expression of two bona fide proliferative markers, PCNA and Ki-67.