Drugs which are commonly-used as the first line of treatment for colorectal cancer cause the tumor cells to oversecrete mucins, according to a new study (“Reversing chemorefraction in colorectal cancer cells by controlling mucin secretion”) published in eLife. The proteins alter the mucous layer, forming a physical barrier that prevents drugs from reaching their intended target.

Using different techniques involving genetic manipulation and the use of chemical inhibitors, a research team in Spain was able to experimentally regulate mucin secretion in cancer cell lines and patient-derived organoids, paving the way for the possible development of future treatments that can be used alongside chemotherapy to target drug-resistant forms of colorectal cancer. The experiments also revealed potential new biomarkers for the prognosis of the disease.

The researchers studied mucins, which are heavy, sugar-coated proteins that are secreted by specialized cells in the linings of the eye, nose, airway, and the digestive tract, including the colon. On average, humans secrete up to a liter of mucins per day. Mucins have gel-forming properties that mix with other biological matter to form mucous, a thick liquid that acts as a lubricant, preventing tissues from becoming dehydrated and also forming a barrier to protect cells from pathogens and other external factors.

Colorectal cancer occurs when tumors develop in the colon or rectum. Adenocarcinomas are the most common type of colorectal cancer, often growing from the cells that constitute the mucous lining of the tissue. The researchers studied whether the overexpression of mucins could create a barrier for drugs reaching the tumor cells.

Experiments with fluorouracil and irinotecan, two drugs given in combination as the first-line treatment for colorectal cancer, revealed that cancer cells respond to the treatment by secreting copious amounts of mucin.

“15% of colorectal cancers (CRC) cells exhibit a mucin hypersecretory phenotype, which is suggested to provide resistance to immune surveillance and chemotherapy. We now formally show that colorectal cancer cells build a barrier to chemotherapeutics by increasing mucins’ secretion,” write the investigators.

“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 subset of untreated tumors. 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 plus Irinotecan (5-FU+iri.) compared to control cells, whereas KChIP3 overexpressing cells are 10 times more sensitive to killing by chemotherapeutics. Similar increase in tumor 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 colorectal cancer cells and other mucinous tumors.”

3d illustration of colon cancer - colon tumor

Colorectal cancer cells build a barrier to chemotherapeutics by increasing mucins’ secretion [Peterschreiber.media/Getty Images]

“What we were surprised to find is that chemotherapy itself triggers mucin secretion, potentially trapping patients in a vicious cycle requiring increasingly higher doses. This can be stopped if chemotherapy is combined with treatments that inhibit mucin secretion,” says ICREA Research Professor Vivek Malhotra, PhD, ICREA Research Professor, coordinator of the cell and developmental biology program at the Center for Genomic Regulation (CRG) at the Barcelona Institute for Science and Technology and author of the study.

The researchers studied different ways of blocking mucin secretion to make tumor cells more sensitive to the drugs. They first attempted to genetically manipulate levels of the protein KChIP3, previously shown by Malhotra’s research group to regulate mucin secretion. They found that colorectal cancer cells depleted of KChIP3 were four times more drug-resistant compared to cells with normal levels of KChIP3. In contrast, cells with higher-than-normal levels of KChIP3 were ten times more sensitive to chemotherapeutic drugs.

According to Lluís Espinosa, PhD, coordinator of the molecular mechanisms of Cancer and stemness the Hospital del Mar Medical Research Institute(IMIM), researcher at CIBER Cancer (CIBERONC), and co-author of the study, “the downside of using KChIP3 as a therapeutic target is that it requires the use of gene therapy, a technology that is still in its developmental stages and also very expensive to use.”

The authors of the study then assessed KChIP3’s potential as a biomarker for the prognosis of colorectal cancer. By studying publicly available datasets following the progression of patients with colorectal cancer patient, they found that high levels of KChIP3 indicated a longer period of time for disease-free survival, the length of time after primary treatment for a cancer ends that the patient survives without any signs or symptoms of that cancer.

Espinosa emphasizes that further work must be carried out to develop KChIP3’s potential as a prognostic biomarker. “One of the main barriers for looking for KChIP3 in clinical settings is that we don’t have the antibodies needed to measure their levels,” he explained. “To include this biomarker in routine practice requires more research to develop ways of measuring it in the first place.”

The researchers studied other methods to block mucin secretion without resorting to genetic manipulation, such as using chemical inhibitors. Previous work by Malhotra’s research group had shown that inhibitors that block the function of sodium and calcium channels, known as NCX blockers, also inhibit the secretion of mucins.

The researchers chose an NCX blocker known as SN-6 for their experiments, which has previously been studied for its potential in treating heart arrhythmias. They tested SN-6 in organoids derived from patients, finding that it made colorectal cancer cells 40 times more sensitive to chemotherapeutic drugs.

According to  Gerard Cantero, PhD, who performed this work at CRG and is now a principal investigator at Vall d’Hebron Research Institute (VHIR), the findings highlight a potential new treatment strategy.

“SN-6 is a highly specific inhibitor, meaning it’s less likely to result in side effects compared to other NCX blockers,” he said. “We found that SN-6 specifically inhibits the secretion of the mucins that are triggered by chemotherapy. Clinical trials are needed to confirm our findings, but combining SN-7 with chemotherapy is of great interest to overcome the side effects of chemotherapy.”

Though this study addressed the problem of colorectal cancer cells, the approach could benefit patients with mucinous cancers or adenocarcinomas in general.

“Mucin is produced in many parts of the body, not just the digestive tract. Further research should explore whether the inhibitors can affect treatment of other types of chemotherapy-resistant cancers that involve a mucous layer,” noted Cantero.

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