Understand how tumor cells respond to a pro-inflammatory microenvironment with
changes in the alternative splicing of RAC1B.FCTN/

Bessa, Cláudia

Gonçalves, Vânia

Jordan, Peter

Matos, Paulo

Pereira, Joana

English

Repositório Científico do Instituto Nacional de Saúde

A pro-inflammatory microenvironment triggers overexpression of tumor-related RAC1B in polarized colorectal cancer cellsJoana F S Pereira, Cláudia Bessa, Vânia Gonçalves, Paulo Matos, Peter JordanDepartamento de Genética Humana,Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugalvania.goncalves@insa.min-saude.ptVirtual Annual Meeting SPGH - 2021IntroductionInflammatory tumor cell microenvironmentRAC1 Gene5’UTR 3’UTR1 2 3 4 5 65’UTR 3’UTR1 2 3 4 5 63bRAC1 TranscriptSwitch I Switch II19a.a.RAC1B TranscriptRAC1B Protein5’UTR 3’UTR57bpSwitch I Switch IIRAC1 Protein• RAC1B stimulates cell survival via NF-𝜅B• Overexpressed in a subgroup of colorectal tumors;• Induced by inflammatory conditions.• Tumor-promoting condition;• Provides survival signals to tumor cells, which respond with changes in gene expression.Alternative splicingCoussens & Werb, Nature, 2002, 420, 860-867; Jordan et al., Oncogene, 1999, 18, 6835-6839; Matos et al., Gastroenterology, 2008, 135, 899-906; Matos et al., Neoplasia, 2013, 15, 102-111Understand how tumor cells respond to a pro-inflammatory microenvironment with changes in the alternative splicing of RAC1BDay 0Day 120 hours48 hoursMeasurement of the transepithelialelectrical resistance (TEER) 24 hoursCRC cellsCaco-2FibroblastsNIH3T3 and CT5.3Immune cellsTHP-1, M0, M1 and M2Experimental procedure:Results: Establishment of a co-culture model between polarized Caco-2 and stromal cellsImmunoblot representative of the protein levels of RAC1B and RAC1 in Caco-2 cell lysates after 48h of co-culture, and the graph corresponds to the respective quantification of relative RAC1B protein levels. Quantification of RAC1Bprotein levels was presented as fold change relative to control (Caco2+Caco-2). Data are means±SEM of 8 experiments; values differing with statistic significance from control: *p<0.05, ***p<0.001; one-way ANOVA with Dunnett’smultiple comparison test.Day 0Day 120 hours48 hoursMeasurement of the transepithelialelectrical resistance (TEER) 24 hoursCRC cellsCaco-2FibroblastsNIH3T3 and CT5.3Immune cellsTHP-1, M0, M1 and M2Experimental procedure:Results: Establishment of a co-culture model between polarized Caco-2 and stromal cellsImmunoblot representative of the protein levels of RAC1B and RAC1 in Caco-2 cell lysates after 48h of co-culture, and the graph corresponds to the respective quantification of relative RAC1B protein levels. Quantification of RAC1Bprotein levels was presented as fold change relative to control (Caco2+Caco-2). Data are means±SEM of 8 experiments; values differing with statistic significance from control: *p<0.05, ***p<0.001; one-way ANOVA with Dunnett’smultiple comparison test.Combined presence of cancer-associated fibroblasts and M1 macrophages induced the most increased levels of RAC1B in Caco-2 cellsHuman Inflammation Antibody Array C3:Human Inflammation Antibody Array C3. Each spot correspond to the presence of one cytokine in the basolateral medium after 48h of co-culture. Immunoblots are representative of the protein levels of RAC1B and RAC1 in Caco-2 celllysates after 48h of treatment with purified cytokines, and the graphs correpond to the quantification of relative RAC1B protein levels. Quantification of RAC1B protein levels was presented as fold change relative to control (Caco-2treated with PBS). Data are means±SEM of 5 experiments; values differing with statistic significance from control: *p<0.05, ***p<0.001; one-way ANOVA with Dunnett’s multiple comparison test.IL-1bIL-6IL-8GM-CSFMIP-2aIL-11Results: Increased levels of cytokines secreted by co-cultured cells associate with increased RAC1BConclusions• Pro-inflammatory signals from the microenvironment modulate RAC1B alternative splicingin Caco-2 colon epithelial cells;• Cytokines IL-1β, IL-6 and IL-8 are increased in conditioned co-culture media of Caco-2+CT5.3+M1, and increased RAC1B levels when added in purified form.AcknowledgementsPeter JordanJoana PereiraCláudia BessaPaulo MatosPatrícia Barros

A pro-inflammatory microenvironment triggers overexpression of tumor-related RAC1B in polarized colorectal cancer cells

http://repositorio.insa.pt/bitstream/10400.18/7975/1/e-poster%20SPGH%202021.pdf

A pro-inflammatory microenvironment triggers overexpression of tumor-related RAC1B in polarized colorectal cancer cells

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