Reversing chemorefraction in colorectal cancer cells by controlling mucin secretion
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Curated by eLife
Evaluation Summary:
The study by Cantero-Recasens et al. aims to investigate if mucus secreted by colorectal cancers would impact the effect of the frequently used chemotherapy treatment, FOLFIRI as it has been reported that mucinous carcinomas are more treatment resistant. They further investigate the role of some mucus secretion regulatory genes in this context. The conclusions made on the effect of the mucus secretion regulatory genes are well supported, though the link to the function of mucus in reducing treatment availability needs some further clarifications.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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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.
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Author Response
Reviewer #1 (Public Review):
The study shows nicely that the calcium binding protein KChiP3 is associated with poor survival of the colorectal cancer (CRC) cohort analyzed and could be a potential prognostic marker. Decreased KChiP3 expression also reduced cell survival upon FOLFIRI treatment making its impact interesting. KChiP3 has previously been indicated by this group to regulate mucus release from the used colon carcinoma cell line and has in other studies been implicated in regulation of calnexin and potassium channels. The inhibitors Benzamil, an Amiloride derivative, and SN-6, a NCX inhibitor, both further reduced survival of colon cancer cells treated with FOLFIRI. These findings reveal an interesting potential of positive effects by manipulating calcium control in the cells to enhance the effect of FOLFIRI …
Author Response
Reviewer #1 (Public Review):
The study shows nicely that the calcium binding protein KChiP3 is associated with poor survival of the colorectal cancer (CRC) cohort analyzed and could be a potential prognostic marker. Decreased KChiP3 expression also reduced cell survival upon FOLFIRI treatment making its impact interesting. KChiP3 has previously been indicated by this group to regulate mucus release from the used colon carcinoma cell line and has in other studies been implicated in regulation of calnexin and potassium channels. The inhibitors Benzamil, an Amiloride derivative, and SN-6, a NCX inhibitor, both further reduced survival of colon cancer cells treated with FOLFIRI. These findings reveal an interesting potential of positive effects by manipulating calcium control in the cells to enhance the effect of FOLFIRI treatment of CRC patients.
The mechanism is speculated to involve modulation of the protective mucus secreted by the cells. The experimental setup is largely based on two subclones of the cancer derived cell line HT29 (M6 and 18N2). These cells produce and secrete different gel-forming mucins, mainly MUC2 and MUC5AC but this could vary depending on confluency, differentiation, and polarization. Is the level affected by these features?
These cells derived from the more heterogeneous HT29 cell line are highly comparable and grow as well-polarized monolayers that differentiate after reaching confluence (Phillips et al., 1995). Thus, all experiments were done on the same day post-confluence. We have now added a specific description of this in the methods section as stated below.
“Cell lines
HT29-M6
HT29-M6 cells (obtained from ATCC) were grown in Dulbecco’s modified Eagle’s medium (Invitrogen) plus 10% foetal bovine serum (Biological Industries) and were maintained in 5% CO2 incubator at 37ºC. All experiments were performed with cells at six days postconfluency when they form a well-polarized monolayer and present higher levels of mucins.
HT29-18N2
HT29-18N2 cells (obtained from ATCC) (RRID:CVCL_5942) were tested for mycoplasma contamination with the Lookout mycoplasma PCR detection kit (Sigma-Aldrich, St. Louis, MO). Mycoplasma negative HT29-18N2 cells were used for the experiments presented here. HT29-18N2 cells were differentiated to goblet cells as described previously (5) Briefly, cells were seeded in complete growth medium (DMEM complemented with 10% FCS and 1% P/S), and the following day (Day zero: D-0), the cells were placed in PFHMII protein free medium (GIBCO, ThermoFisher Scientific, Waltham, MA, USA). After 3 days (D-3), medium was replaced with fresh PFHMII medium and cells grown for 3 additional days. At day 6 (D-6) cells were trypsinized and seeded for the respective experiments in complete growth medium followed by incubation in PFHMII medium at day 7 (D-7). All experimental procedures were performed at day 9 (D-9).”
The inhibitory effect of mucus was addressed by the studying the effect of FOLFIRI treatment on MUC5AC production and secretion. The initial experiments showed increased production of MUC5AC using western blot analysis with a peak intensity of the protein after three hours of FOLFIRI treatment. The band, as determined by the size, corresponds to the non-glycosylated newly produced mucin product from the endoplasmic reticulum.
We apologize for this misunderstanding. The highest molecular weight marker (MWM) that we have runs at 450 kDa, which we erroneously indicated 560 kDa in the figure. The molecular weight of MUC5AC is 560 kDa. The anti- MUC5AC antibody detects a smear at molecular weights higher than 450 kDa and migrate at the top of a 4% acrylamide gel. We have now indicated the size of the 450 kDa MWM (Figure 1A, Figure 4A). The detected bands therefore do not correspond to newly produced mucins. We have now confirmed the presence of mature mucins in our cells and also PDOs by Alcian Blue staining (Figure 4 – Supplement figure 1A and 1C).
These results are supplemented with detection of increased intracellular intensity in cells stained for MUC5AC, but these findings do not seem to be reproduced in the following control experiments studying effects of Benzamil or SN-6.
In Figure 4A we also detected an increase in MUC5 levels at 3 hours of 5-FU+Iri. treatment. However, due to the high levels of MUC5AC in the SN-6 adjacent lanes it is difficult to visualize this increase. We have now repeated the experiment to obtain better and clearer images (Figure 4A).
The amounts of transcriptionally expressed or mature secreted mucus are however not determined upon FOLFIRI treatment nor in the experiments manipulating mucus secretion by altered KChiP3 expression or inhibited NCX function. In this context the extracellular mucus could be a combination of truly secreted mucus and intracellular components from detached or dead cells, but how this contributes to cell survival is not known.
Proper controls for all experiments involving measurements in the extracellular medium were performed to ensure that secreted medium did not contain dead or detached cells. We now include a western blot of the secreted medium against cytosolic marker, which shows there are no dead cells in the experiments (Figure 4A). In addition, we have tested MUC5AC RNA levels in HT29 cells treated with FOLFIRI, SN-6 and SN-6+FOLFIRI (Figure 4 – Supplement figure 1B). These results confirm that there is no transcriptional effect on mucins.
The inhibitor effect of mucus on treatment of CRC cells is speculated to be though binding of the drugs to the mucus. This is studied indirectly by binding of albumin to cells with or without secreted mucus. Mucus binding is however influenced by several factors as mucus composition, organization, and environmental impacts and as the binding affinity is dependent on the molecule the difference between albumin and FOLFIRI is a concern.
We apologize for not explaining this clearly. We propose that mucins act as a physical barrier for 5-FU internalization and we have used albumin as a means to show the effect on internalization by excessive extracellular mucins. The possibility that 5-FU+iri. is actively retained by binding to mucin fibers is a possibility that we have not addressed. This requires the availability of tagged therapeutics for visualization by fluorescence microscopy and currently unavailable. However, this is an important issue and should be investigated in the future. We now include this possibility in the discussion as stated below:
“Cancer cells produce mucins as a protective response to chemotherapy
Our data show that CRC cells produce mucins in response to 5-Fluorouracil + Irinotecan (5-FU+iri.), a first-choice chemotherapy for most CRC (30). Besides, 5-FU+iri. not only increases mucin production, but also increases mucin secretion (70-80% after 6 hours). This increase in secretion is completely blocked by SN-6 treatment (as described previously in (6)). This is extremely important, because secreted mucins can create a physical barrier that could prevent drugs (for instance, chemotherapeutics) from reaching the tumour cells. Another possibility is that 5-FU+iri. is actively retained by mucin fibres. The availability of tagged versions of these chemicals will help to address this issue in the future.”
The provided images also indicate mucus free areas that could allow for compound access if not covered by other unstained mucins. Organoids were used to assess the effect of SN-6 which also in this system reduce cell survival combined with FOLFIRI treatment but the link to mucus secretion was not explored.
We provide now additional staining with Alcian Blue from HT-29 and PDOs cultures (Figure 4 – Supplement figure 1A and 1C).
Taken together the basis for the arguments that mucus is influencing the accessibility of the supplemented drugs is not fully supported by the experiments provided.
As stated above, we now provide new data to clarify the effect of mucus on chemoresistance (Figure 4A, Figure 4 – Supplement figure 1A and 1C). We also explain this more clearly in the discussion as stated below.
“Cancer cells produce mucins as a protective response to chemotherapy
Our data show that CRC cells produce mucins in response to 5-Fluorouracil + Irinotecan (5-FU+iri.), a first-choice chemotherapy for most CRC (30). Besides, 5-FU+iri. not only increases mucin production, but also increases mucin secretion (70-80% after 6 hours). This increase in secretion is completely blocked by SN-6 treatment (as described previously in (6)). This is extremely important, because secreted mucins can create a physical barrier that could prevent drugs (for instance, chemotherapeutics) from reaching the tumour cells. Another possibility is that 5-FU+iri. is actively retained by mucin fibres. The availability of tagged versions of these chemicals will help to address this issue in the future. Our results in PDOs and cell lines show that cancer cells respond to chemotherapeutics by secreting copious amounts of mucin to form a barrier against the treatment. We suggest that this reaction (mucin secretion) of colorectal cancer cells to chemotherapy is similar to the programmed response of epithelial cells to toxics or pathogens. In this situation, which could resemble the treatment with 5-FU+iri., mucin-producing cells would release large quantities of mucins to isolate them from the insults (toxins, allergens or pathogens) (3). An interesting question is how 5-FU+iri. triggers mucin secretion and whether there is a specific receptor involved in this response or an intracellular pathway that is activated by these chemicals. This finding is important in application of chemotherapeutics and merits further investigation. Our results show that this increase in mucin production/secretion triggered by chemotherapeutics is independent of transcriptional effects on mucins, although we cannot completely discard the possibility that the levels of components necessary for mucin modification and secretion (e.g., glycosyltransferases) are altered.”
The CRC survival analysis shows that MUC5AC expression levels were not found to be associated with disease outcome but the influence of other mucins was not analyzed. Some previous studies suggest that MUC2 expression is associated with better survival while MC5AC show opposite effects.
As suggested by the reviewer, we have checked the effect of MUC2 on patients’ survival (Figure 2 – Supplement figure 1B). Our analysis revealed that patients with low levels of MUC2 (possibly because cells are more dedifferentiated) have worse prognosis. However, when we stratify patients with high MUC2 levels by KChIP3 expression, we found a tendency (p=0.08) of patients with low levels of KChIP3 to have lower disease-free survival (Figure 2 – Supplement figure 1C). It is interesting to note that patients that had worse prognosis with low levels of MUC5AC and KChIP3 (Figure 2C), had high levels of other secreted mucins like MUC2, MUC4 or MUC5B (Figure 2 – Supplement figure 1A), which could compensate for the lack of MUC5AC. We include this new data in the results section as stated below.
“There is no significant effect on patients with low MUC5AC expression (Figure 2C), although after 100 months there is a decrease in DFS. Further analysis of this effect revealed that three patients (GSM358532, GSM358534 and GSM358438) were responsible of this effect. We studied these patients in more detail and interestingly, although they have low levels of MUC5AC, these patients present increased levels of other secreted mucins (e.g., MUC2) (Figure 2 – Supplement figure 1A). However, similarly to previous studies (10, 12), analysis of the effect of MUC2 levels on DFS shows that high MUC2 levels are protective while patients with low levels of MUC2, which may reflect a more dedifferentiated state of cancer cells, have a lower DFS (HR=2.54, p=0.023) (Figure 2 – Supplement figure 1B). Importantly, when we studied the effect of KChIP3 on the DFS of patients with high expression of MUC2, we found a clear tendency (p=0.08) to worse prognosis in patients with low levels of KChIP3 (Figure 2 – Supplement figure 1C). Altogether, these results suggest that low levels of KChIP3, which consequently increase the quantity of mucins secreted, protect cancer cells from chemotherapeutic drugs.”
As the analysis do not link mucus secretion to treatment efficacy combined with analyses of a role for other regulatory proteins it is not clear how this adds to the aim of the study.
We now show the levels of secreted mucins (Figure 4A), which strengthens the relationship between inhibition of proteins that regulate mucin secretion and chemotherapy efficacy.
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Evaluation Summary:
The study by Cantero-Recasens et al. aims to investigate if mucus secreted by colorectal cancers would impact the effect of the frequently used chemotherapy treatment, FOLFIRI as it has been reported that mucinous carcinomas are more treatment resistant. They further investigate the role of some mucus secretion regulatory genes in this context. The conclusions made on the effect of the mucus secretion regulatory genes are well supported, though the link to the function of mucus in reducing treatment availability needs some further clarifications.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
The study shows nicely that the calcium binding protein KChiP3 is associated with poor survival of the colorectal cancer (CRC) cohort analyzed and could be a potential prognostic marker. Decreased KChiP3 expression also reduced cell survival upon FOLFIRI treatment making its impact interesting. KChiP3 has previously been indicated by this group to regulate mucus release from the used colon carcinoma cell line and has in other studies been implicated in regulation of calnexin and potassium channels. The inhibitors Benzamil, an Amiloride derivative, and SN-6, a NCX inhibitor, both further reduced survival of colon cancer cells treated with FOLFIRI. These findings reveal an interesting potential of positive effects by manipulating calcium control in the cells to enhance the effect of FOLFIRI treatment of CRC …
Reviewer #1 (Public Review):
The study shows nicely that the calcium binding protein KChiP3 is associated with poor survival of the colorectal cancer (CRC) cohort analyzed and could be a potential prognostic marker. Decreased KChiP3 expression also reduced cell survival upon FOLFIRI treatment making its impact interesting. KChiP3 has previously been indicated by this group to regulate mucus release from the used colon carcinoma cell line and has in other studies been implicated in regulation of calnexin and potassium channels. The inhibitors Benzamil, an Amiloride derivative, and SN-6, a NCX inhibitor, both further reduced survival of colon cancer cells treated with FOLFIRI. These findings reveal an interesting potential of positive effects by manipulating calcium control in the cells to enhance the effect of FOLFIRI treatment of CRC patients.
The mechanism is speculated to involve modulation of the protective mucus secreted by the cells. The experimental setup is largely based on two subclones of the cancer derived cell line HT29 (M6 and 18N2). These cells produce and secrete different gel-forming mucins, mainly MUC2 and MUC5AC but this could vary depending on confluency, differentiation, and polarization. The inhibitory effect of mucus was addressed by the studying the effect of FOLFIRI treatment on MUC5AC production and secretion. The initial experiments showed increased production of MUC5AC using western blot analysis with a peak intensity of the protein after three hours of FOLFIRI treatment. The band, as determined by the size, corresponds to the non-glycosylated newly produced mucin product from the endoplasmic reticulum. These results are supplemented with detection of increased intracellular intensity in cells stained for MUC5AC, but these findings do not seem to be reproduced in the following control experiments studying effects of Benzamil or SN-6. The amounts of transcriptionally expressed or mature secreted mucus is however not determined upon FOLFIRI treatment nor in the experiments manipulating mucus secretion by altered KChiP3 expression or inhibited NCX function. In this context the extracellular mucus could be a combination of truly secreted mucus and intracellular components from detached or dead cells, but how this contributes to cell survival is not known. The inhibitor effect of mucus on treatment of CRC cells is speculated to be though binding of the drugs to the mucus. This is studied indirectly by binding of albumin to cells with or without secreted mucus. Mucus binding is however influenced by several factors as mucus composition, organization, and environmental impacts and as the binding affinity is dependent on the molecule the difference between albumin and FOLFIRI is a concern. The provided images also indicate mucus free areas that could allow for compound access if not covered by other unstained mucins. Organoids were used to assess the effect of SN-6 which also in this system reduce cell survival combined with FOLFIRI treatment but the link to mucus secretion was not explored. Taken together the basis for the arguments that mucus is influencing the accessibility of the supplemented drugs is not fully supported by the experiments provided.
The CRC survival analysis shows that MUC5AC expression levels were not found to be associated with disease outcome but the influence of other mucins was not analyzed. Some previous studies suggests that MUC2 expression is associated with better survival while MC5AC show opposite effects. As the analysis do not link mucus secretion to treatment efficacy combined with analyses of a role for other regulatory proteins it is not clear how this adds to the aim of the study.
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Reviewer #2 (Public Review):
The main goal of this work is to test the hypothesis that (a) secreted mucins act as a barrier to protect CRC cells from toxic drugs, and (b) the level of KChIP3 is a determinant of mucin secretion and therefore of chemoresistance in CRC. Experiments with cultured CRC cells and organoids provide solid support for the hypothesis. Apart from some suggestions about presentation as indicated below, the science is persuasive, and it is likely to prove useful for developing improved treatment options for CRC and other mucinous tumors.
Perhaps the most interesting biological question, touched on but not answered in the manuscript, is why chemotherapeutic drugs increase mucin secretion in CRC cells. The implication is that CRC cells have a built-in resistance pathway for toxic drugs, but it is hard to see how such a …
Reviewer #2 (Public Review):
The main goal of this work is to test the hypothesis that (a) secreted mucins act as a barrier to protect CRC cells from toxic drugs, and (b) the level of KChIP3 is a determinant of mucin secretion and therefore of chemoresistance in CRC. Experiments with cultured CRC cells and organoids provide solid support for the hypothesis. Apart from some suggestions about presentation as indicated below, the science is persuasive, and it is likely to prove useful for developing improved treatment options for CRC and other mucinous tumors.
Perhaps the most interesting biological question, touched on but not answered in the manuscript, is why chemotherapeutic drugs increase mucin secretion in CRC cells. The implication is that CRC cells have a built-in resistance pathway for toxic drugs, but it is hard to see how such a pathway could have evolved specifically to promote chemoresistance. A possible explanation is that increased mucin secretion is a general stress response in certain cell types, both normal and cancerous. Experiments to explore this concept would enhance the paper by linking cell biology to potential clinical approaches.
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