Reversing chemorefraction in colorectal cancer cells by controlling mucin secretion

Abstract

Fifteen percent of colorectal cancer (CRC) cells exhibit a mucin hypersecretory phenotype, which is suggested to provide resistance to immune surveillance and chemotherapy. We now formally show that CRC cells build a barrier to chemotherapeutics by increasing mucins' secretion. We show that low levels of KChIP3, a negative regulator of mucin secretion (Cantero-Recasens et al., 2018), is a risk factor for CRC patients' relapse in a subset of untreated tumours. Our results also reveal that cells depleted of KChIP3 are four times more resistant (measured as cell viability and DNA damage) to chemotherapeutics 5-fluorouracil + irinotecan (5-FU+iri.) compared to control cells, whereas KChIP3-overexpressing cells are 10 times more sensitive to killing by chemotherapeutics. A similar increase in tumour cell death is observed upon chemical inhibition of mucin secretion by the sodium/calcium exchanger (NCX) blockers (Mitrovic et al., 2013). Finally, sensitivity of CRC patient-derived organoids to 5-FU+iri. increases 40-fold upon mucin secretion inhibition. Reducing mucin secretion thus provides a means to control chemoresistance of mucinous CRC cells and other mucinous tumours.

Keywords: 5-FU+iri.; KChIP3; cell biology; chemoresistance; chemotherapy; colorectal cancer; human; mucins.

Figure 1.. Mucins in colorectal cancer.

(A) Cell lysates from differentiated HT29-M6 cells treated with 5-fluorouracil + irinotecan (5-FU + iri.) (50 µg/mL 5-FU + 20 µg/mL iri.) for 24 hr were analysed by Western blot with an anti-MUC5AC to test expression levels. Tubulin was used as a loading control. (B) Immunofluorescence Z-stack projections of differentiated HT29-M6 cells treated with vehicle (control) or 5-FU + iri. (50 µg/mL 5-FU + 20 µg/mL irinotecan) for 24 hr. Cells were stained with anti-MUC5AC (red) and DAPI (blue). Scale bar = 50 µm. (C) Differentiated HT29 cells were treated with 100 µM ATP for 30 min, washed extensively (upper panel) or smoothly (lower panel), and incubated with Alexa Fluor 488-labelled albumin for 1 hr. Cells were stained with anti-MUC5AC antibody (red) and DAPI (blue). In the orthogonal view, dotted lines across the images demarcate the top surface of the cell. Scale bars correspond to 5 µm. (D) Colocalization between MUC5AC and albumin was calculated from immunofluorescence images by Manders’ coefficient using Fiji. Average values ± SEM are plotted as scatter plot with bar graph. The y-axis represents Manders’ coefficient of the fraction of albumin overlapping with MUC5AC (N ≥ 3). (E) Consensus molecular subtype (CMS) distribution of high and low MUC5AC-expressing colorectal tumours (Jorissen cohort). (F) Disease-free survival (DFS) of colorectal cancer patients with high (n = 73) or low (n = 153) MUC5AC levels (Jorissen cohort). NOLBL: samples without a defined CMS subtype (tumours with no label). *p<0.05, **p<0.01.

Figure 1—figure supplement 1.. Experimental design for measuring mucin production after treatment.

(A) Schematic diagram of the experimental set-up to measure mucin production after 5-fluorouracil + irinotecan (5-FU + iri.) treatment.

Figure 2.. KChIP3 is a prognostic marker of colorectal cancer (CRC).

(A–C) Disease-free survival (DFS) according to KChIP3 levels of CRC patients (low KChIP3 levels, n = 120; high KChIP3 levels, n = 106) (A), CRC patients with high MUC5AC levels (low KChIP3 levels, n = 39; high KChIP3 levels, n = 34) (B), or CRC with low MUC5AC levels (low KChIP3 levels, n = 81; high KChIP3 levels, n = 72) (C). (D) Differentiated control (black), KChIP3-overexpressing cells (KCNIP3-OV, blue) and KChIP3-depleted cells (KCNIP3-KD, red) were treated for 72 hr with increasing concentrations of 5-fluorouracil + irinotecan (5-FU + iri.). Average values ± SEM are plotted as scatter plot (N > 3). The y-axis represents the percentage of cell growth relative to the lowest concentration of 5-FU + iri. The IC50 was calculated from the interpolated curve. HR, hazard ratio. *p<0.05, **p<0.01.

Figure 2—figure supplement 1.. Expression of mucins in patients.

(A) Expression levels of secreted mucins MUC2, MUC5AC, MUC5B, MUC6, MUC7, and MUC19. Each dot represents a different patient. Patients GSM358532, GSM358535, and GSM358538 are highlighted in green, blue, and red, respectively. (B) Disease-free survival (DFS) of colorectal cancer patients with high (n = 73) or low (n = 153) MUC2 levels (Jorissen cohort). (C) DFS according to KChIP3 levels of colorectal cancer patients with high MUC2 expression (low KChIP3 levels, n = 27; high KChIP3 levels, n = 30).

Figure 3.. Inhibition of sodium/calcium exchangers (NCXs) enhances cell death by 5-fluorouracil + irinotecan (5-FU+ iri.).

(A) Comet assay of HT29-M6 cell line treated with 5-FU + iri. (25 µg/mL 5-FU + 10 µg/mL irinotecan) and 20 µM benzamil, alone or in combination. The tail moment was measured after 72 hr of treatment (N > 3). (B) Quantification of cell viability in HT29-M6 cell line after treatment (24 or 72 hr) with 5-FU + iri. (10 µg/mL 5-FU + 4 µg/mL Irinotecan) and 20 µM benzamil, alone or in combination (N ≥ 3). (C) Immunofluorescence Z-stack projections of differentiated HT29-M6 cells treated with vehicle, 20 µM benzamil, or 10 µM SN-6 in the presence or absence of 5-FU + iri. Cells were stained with anti-MUC5AC (green), phalloidin (red), and DAPI (blue). Scale bar = 5 µm. (D) Quantification of the number (upper graph) and volume (lower graph) of MUC5AC granules from immunofluorescence images by confocal microscope. Average values ± SEM are plotted as scatter plot with bar graph (N ≥ 3). Veh., vehicle; Benz., benzamil. *p<0.05, **p<0.01.

Figure 4.. SN-6 treatment increases sensitivity of colorectal cancer (CRC)-derived cells and organoids to 5-fluorouracil + irinotecan (5-FU + iri.).

(A) Cell lysates and secreted medium of differentiated HT29-M6 cells pre-treated with a vehicle or 10 µM SN-6 inhibitor for 24 hr and then exposed to 5-FU + iri. (50 µg/mL 5-FU + 20 µg/mL iri.) for 0, 1, 3, 6, and 24 hr were analysed by Western blot with an anti-MUC5AC, anti-γH2A.X, and H3 to test their levels. Tubulin was used as a loading control. Quantification of MUC5AC and γH2A.X is included (N ≥ 3). (B) Immunofluorescence images of differentiated HT29-M6 cells treated with vehicle (control) or 10 µM SN-6 in the presence or absence of 5-FU + iri. (50 µg/mL 5-FU + 20 µg/mL irinotecan) for 24 hr. Cells were stained with anti-MUC5AC (red), anti-γH2A.X (green), and DAPI (blue). Scale bar = 50 µm. (C) Quantification of the number of γH2A.X-positive cells in the different conditions relative to the total number of cells from the immunofluorescence images (N ≥ 3). (D) CRC patient-derived organoids (PDOs) were treated for 72 hr with increasing concentrations of 5-FU + iri. with vehicle or 10 µM SN-6. Average values ± SEM are plotted as scatter plot. The y-axis represents the percentage of cell growth relative to the lowest concentration of 5-FU + iri. The IC50 was calculated from the interpolated curve (N ≥ 3). *p<0.05, **p<0.01.

Figure 4—figure supplement 1.. Mucins’ levels in HT29 cells and in a patient-derived organoid.

(A) Schematic diagram of the experimental set-up to measure mucin secretion and production. (B) Representative images of HT29-M6 differentiated cell (vehicle [control], 5-fluorouracil + irinotecan [5-FU+ iri.], SN-6, and 5-FU + iri. + SN-6) stained with Alcian blue. Scale bar = 50 µm. (C) Relative MUC5AC RNA levels in HT29-M6 treated with vehicle (control), 5-FU + iri., SN-6, and 5-FU + iri. + SN-6 (N = 3). (D) Alcian blue-stained patient-derived organoid to visualize external mucins (in blue). Scale bar = 50 µm.