Lactobacillus reuteri decreases adherent tumor mucins and enhances chemotherapeutic susceptibilities via ERK/JNK pathways
Authors: Engevik Melinda Anne , Luk Berkley , Hall Anne , Ganesh Bhanu Priya , Versalovic James 
Published: November 2023

BackgroundColorectal cancer (CRC) is the second leading cause of cancer related deaths in the United States. Although CRC is commonly treated with the chemotherapeutict Fluorouracil (5‐FU), only 10–15% of advanced CRC tumors respond. Mucins, a hallmark of carcinomas derived from epithelia, have long been implicated in the pathogenesis of cancer including CRC. The cancer mucin complex created by adherent mucins (MUC1, MUC3, MUC4) and secreted mucins (MUC2, MUC5AC) have been hypothesized to limit the uptake of the chemotherapy drugs, thus promoting chemoresistance. Several members of the gut microbiota are capable of altering mucin production and may represent potential treatments to increase the chemotherapeutic susceptibility in resistant cancers.Methods & ResultsThe mucus producing adenocarcinoma cell line HT‐29‐MTX‐E12, which aberrantly expresses both adherent and secreted mucins, was incubated with conditioned media (CM) from probiotic human gut microbes. Analysis of MUC expression by qRT‐PCR revealed that several Bifidobacterium spp. and Lactobacillus spp. were capable of decreasing adherent and secreted mucin expression. Of the Lactobacillus members, L. reuteri ATCC 6475 was capable of altering total adherent mucins, as determined by immunofluorescence (IF), alcian blue (AB), and biotinylation. L. reuteri was able to stimulate expulsion of stored mucin granules as determined by media AB and MUC5AC protein as determined by IF. To assess whether bacterial‐specific changes in mucin expression correlated with increased cancer susceptibility to drug treatment, HT‐29‐MTX‐E12 cells were incubated with CM followed by the chemotherapeutic agent 5‐FU. L. reuteri CM increased susceptibility to 5‐FU treatment resulting in significant decreases in cancer cell viability as determined by resazurin. This effect was independent of lactate. The L. reuteri secreted factor was found to be a >10 kDa, heat stable protein. 130 L. reuteri mutants were screened from a secretome library and three mutants were identified that lacked the ability to promote 5‐FU susceptibility. Two mutants encoded uncharacterized proteins, and one mutant encoded a bacterial ABC transporter. Magpix multiplex assay revealed that WT L. reuteri, but not the mutants, were able to promote ERK, CREB, and JNK phosphorylation. No changes were observed in p7056, STAT3, STAT5, p38 or NF‐κB. This correlated with no alteration in 5‐FU susceptibility when the cells were pre‐treated with an NF‐κB inhibitor. To establish the ability of L. reuteri to alter mucins and promote tumor 5‐FU susceptibility in vivo, C57BL/6J‐ApcMin/J mice were colonized with L. reuteri bacteria or treated with un‐inoculated bacterial media. L. reuteri colonized ApcMin mice had significantly decreased adherent Muc1, Muc4, and Muc5ac tumor masses compared with media‐treated mice, consistent with the HT29‐MTX cell line data. To examine 5‐FU susceptibility ApcMin mouse tumors were excised and ex‐vivo treated with L. reuteri CM followed by 5‐FU. Addition of CM resulted in enhanced susceptibility of male and female intestinal tumors to 5‐FU treatment as assessed by resazurin.ConclusionTogether this data suggests that bacterial‐induced modifications of cancer‐driven mucus in combination with traditional chemotherapeutic agents may provide new therapeutic strategies for the treatment of chemoresistant cancer.Support or Funding InformationT32 DK007664