Reversing chemorefraction in colorectal cancer cells by controlling mucin secretion

  1. Gerard Cantero-Recasens  Is a corresponding author
  2. Josune Alonso-Marañón
  3. Teresa Lobo-Jarne
  4. Marta Garrido
  5. Mar Iglesias
  6. Lluis Espinosa  Is a corresponding author
  7. Vivek Malhotra  Is a corresponding author
  1. Renal Physiopathology Group, Vall d’Hebron Research Institute (VHIR), Spain
  2. Cancer Research Program, Institut Mar d'Investigacions Mèdiques, CIBERONC Hospital del Mar, Spain
  3. Department of Pathology, Institut Mar d'Investigacions Mèdiques, Universitat Autònoma de Barcelona, CIBERONC, Spain
  4. Centre for Genomic Regulation (CRG), The Barcelona Institute for Science and Technology, Spain
  5. Institució Catalana de Recerca i Estudis Avançats (ICREA), Spain
4 figures, 2 tables and 1 additional file

Figures

Figure 1 with 1 supplement
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 …

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 with 1 supplement
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 …

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, …

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 …

Figure 4 with 1 supplement
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 …

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 …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Homo sapiens)MUC5ACEnsemblENSG00000215182
Gene (H. sapiens)MUC2EnsemblENSG00000198788
Gene (H. sapiens)MUC6EnsemblENSG00000184956
Gene (H. sapiens)MUC5BEnsemblENSG00000117983
Gene (H. sapiens)MUC19EnsemblENSG00000205592
Gene (H. sapiens)KCNIP3EnsemblENSG00000115041
Cell line (H. sapiens)HT29-M6ATCCCVCL_G077Mycoplasma free
Cell line (H. sapiens)HT29-18N2ATCCCVCL_5942Mycoplasma free
AntibodyAnti-MUC5AC (mouse monoclonal)Neomarkers,Waltham, MAClone 45M1(1:1000)
AntibodyAnti-γH2A.X (mouse monoclonal)Cell Signaling#2577(1:1000)
Commercial assay or kitCometAssay Trevigen KitTrevigen250-050K
Chemical compound, drugSN-6Sigma-AldrichSML1937-5MG(5 µM)
Chemical compound, drugBenzamilSigma-AldrichB2417-10MG5 µM
Table 1
Primer sequences used for detecting mRNA for the respective genes.
GeneForward primer (5′–3′)Reverse primer (5′–3′)
MUC5ACCTGGTGCTGAAGAGGGTCATCAACCCCTCCTACTGCTACG
TBPTGCCCGAAACGCCGAATATAATCGTCTGGACTGTTCTTCACTCTTGG
GAPDHGTCATCCCTGAGCTGAACGCTCCTTGGAGGCCATGTG

Additional files

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