- Email: [email protected]
The Myofibroblasts’ War on Drugs
Developmental Cell
Previews The Myofibroblasts’ War on Drugs Jens Puschhof1,2 and Hans Clevers1,2,3,* 1Hubrecht
Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Uppsalalaan 8, 3584 CT Utrecht, the Netherlands Institute, Utrecht, the Netherlands 3Princess Ma ´ xima Centre, 3584 CT Utrecht, the Netherlands *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2018.09.008 2Oncode
The intestinal lining faces a constant assault from harmful gut contents. A study by Chee and colleagues in this issue of Developmental Cell describes how a population of myofibroblasts resists these threats and boosts regeneration of the intestinal epithelium. The human intestinal tract is constantly exposed to a plethora of potentially dangerous substances, ranging from poisonous compounds in food to bacterial metabolites and orally administered drugs. The epithelium lining the intestine is the first barrier against these compounds, and it copes with the incurred damage by active expulsion of small molecules and by renewing itself every 5 days. This renewal is driven by stem cells residing in intestinal crypts and supported by Wnt-producing epithelial Paneth cells. Another crucial source of Wnt niche signals are myofibroblasts located in the mesenchyme beneath the epithelium (Roulis and Flavell, 2016). Although these subepithelial myofibroblasts are exposed to similar threats from damaging luminal contents, they do not regenerate as quickly as the epithelium. To date, myofibroblasts’ mechanisms for coping with these insults have not been elucidated. A study in this issue of Developmental Cell (Chee et al., 2018) offers a compelling explanation for how the intestinal stroma resists hazardous intestinal contents. The study finds that a subpopulation of the myofibroblasts is intrinsically resistant to several xenobiotics and avoids excessive damage due to the expression of specific drug efflux pumps (Figure 1). The authors start by investigating a surprising discrepancy: shutting down Wnt ligand secretion through PORCN inhibition or genetic knockout in purely epithelial ‘‘mini-guts’’ (organoids) leads to complete disruption of the stem cell niche. In striking contrast, mice treated with comparable doses of PORCN inhibitors display negligible gut toxicity (Madan et al., 2016), and an early-stage clinical trial corroborates these findings in hu-
mans (Ng et al., 2017). The authors propose that the explanation for this is found in the myofibroblasts, as the sensitivity of epithelial organoids to PORCN inhibition was reduced when they were co-cultured with myofibroblasts. Previous reports had indeed found that non-epithelial Wnt sources stabilize the stem cell niche upon epithelial Porcn knockout (San Roman et al., 2014). Chee et al. (2018) found that co-culture with other Wnt-producing cells was not able to rescue PORCN inhibition-induced toxicity in intestinal organoids, hinting at a myofibroblast-specific resistance to PORCN inhibition. This observation prompted the authors to ask whether these myofibroblasts might also show an insensitivity to other xenobiotics. To answer this question and to investigate resistance to tamoxifen-induced recombination, the authors employed a Rosa26CreERT2;mT/mG mouse model. In this mouse model, all cells express membrane targeted tandem-dimer tomato (mT), which gets switched to membrane targeted green fluorescent protein (mG) upon tamoxifen-induced Cre activation, making it a useful tool to study global Cre activity. Consistent with the authors’ hypothesis, a subpopulation of subepithelial myofibroblasts did not switch color in response to tamoxifen, regardless of timings and route of administration, indicating that tamoxifen was not able to act in this population. The tamoxifen-resistant myofibroblasts were identified through expression of a-SMA and, in some cases, PDGFRa. In contrast, other tissues such as the liver showed recombination of the mT/mG allele in all cells and thus a complete tamoxifen response. Chee et al. (2018) go on to show that the tamoxifen-resistant population of myofibroblasts is also insensitive to the
anthracycline chemotherapeutic doxorubicin, highlighting its resistance to multiple xenobiotic agents. To determine whether the resistance to xenobiotics observed in this population of myofibroblasts was due to drug export from the cells, the authors used RNA sequencing to profile multidrug resistance transporter (MDR) expression in myofibroblasts. Intriguingly, Chee et al. (2018) found a subset of drug transporters including the ATP-binding cassette (ABC) transporters Abcc1 and Abcb1b overexpressed in myofibroblasts compared to epithelial crypts. In a set of elegant experiments, the authors identify ABCC1 as the dominant transporter relevant to myofibroblast resistance to PORCN inhibition. First, overexpression of ABCC1 in HEK293 cells increased expulsion of fluorescently labeled PORCN inhibitors. Second, specific inhibition of ABCC1 led to increased retention of PORCN inhibitor in myofibroblasts. Inhibition of ABCB1 had a smaller effect. These results are in line with experiments showing that while the intestines of Abcc1 KO and Abcb1 KO mice treated with PORCN inhibitors both displayed decreased expression of Wnt target genes as compared to WT controls, the difference was most notable in the Abcc1 KO intestines. Finally, ABCC1 inhibition also supported tamoxifen-induced recombination in the previously resistant myofibroblast population in vivo. Looking beyond the small intestine, the authors observe tamoxifen-resistant cell populations in organs such as the large intestine, lung, stomach, kidneys, and pancreas, but not in other tissues like liver and skeletal muscle. A more detailed characterization of these cell types will be essential to assessing their impact on
Developmental Cell 46, September 24, 2018 ª 2018 Elsevier Inc. 669
Developmental Cell
Previews
As PORCN inhibitors are currently being tested in clinical trials in patients with several types of cancers and ABC transporter inhibitors are being evaluated in combination with other anti-cancer therapies, this study has further important implications: inhibition of drug pumps in myofibroblasts may severely potentiate the gastrointestinal toxicity of compounds. Careful consideration of the differential resistance mechanisms of cancer cells and the intestinal stem cell niche may however yield viable strategies to selectively boost efficacy of anti-cancer drugs.
REFERENCES
Figure 1. Myofibroblasts Provide a Xenobiotic-Resistant Stem Cell Niche through ABCC1 Transporter Expression Xenobiotics from the intestinal lumen harm the epithelial cells in the crypt but are actively expelled from subepithelial myofibroblasts through ABCC1 transporters. Epithelial Paneth cells stop secreting Wnt ligands in response to some xenobiotics, which is rescued by myofibroblast-derived Wnt ligands. Image adapted from Servier Medical Art (https://smart.servier.com/).
lineage tracing studies in these organs and to forming an understanding of their role in response to damaging agents. The results presented by Chee et al. (2018) require that the field use caution when interpreting the results of previous reports using chemically induced activation and recombination of fibroblasts. In a previous study, a tamoxifen-induced simultaneous epithelial and subepithelial knockout of Porcn did not induce major gut disruption (San Roman et al., 2014). The new results suggest that these experiments did not yield a phenotype because of a tamoxifen-resistant population of myofibroblasts that did not lose Porcn expression and was thus able to continue providing the stem cell niche. For the large intestine, a recent study (Degirmenci et al., 2018) describes an essential Wnt ligand producing mesenchymal population marked by GLI1 and PDGFRa. This population was identified through tamoxifen-induced recombination and therefore appears to be sensitive to this xenobiotic. These findings highlight the need for a more detailed characterization of the resistant myofibroblast population in the small intestine, contrasting it to other xenobiotic-sensitive mesenchymal cell populations. The findings presented provide a new angle on the response of the intestinal stem cell niche under xenobiotic stress.
The importance of Abcc1 expression for myofibroblast resistance to several compounds is convincingly demonstrated. While the resistance to tamoxifen and PORCN inhibitors is highly relevant to genetic studies and translational efforts, more nuanced investigations will be necessary to elucidate the cell-type-specific role of individual ABC transporters in response to natural compounds. In particular, considering bacterial metabolites could yield important translational insights, as an Abcc1 / model showed increased susceptibility toward developing inflammatory bowel disease (IBD) (ten Hove et al., 2002). Since the epithelium is even more directly exposed to luminal compounds, it is of high interest to understand why only myofibroblasts would express the described subset of ABC transporters. The authors have also identified a set of ABC transporters enriched in epithelial cells versus myofibroblasts, which adds more detail to previous reports (Leslie et al., 2005; Peng et al., 1999). Further studies on the differential response to hazardous compounds between epithelium and mesenchyme may shed light on specialized resistance mechanisms in both compartments and provide insights as to how niche signals beyond Wnt ligands are provided under xenobiotic stress conditions.
670 Developmental Cell 46, September 24, 2018
Chee, Y.C., Pahnke, J., Bunte, R., Adsool, V.A., Madan, B., and Virshup, D.M. (2018). Intrinsic xenobiotic resistance of the intestinal stem cell niche. Dev. Cell 46, this issue, 681–695. Degirmenci, B., Valenta, T., Dimitrieva, S., Hausmann, G., and Basler, K. (2018). GLI1-expressing mesenchymal cells form the essential Wntsecreting niche for colon stem cells. Nature 558, 449–453. Leslie, E.M., Deeley, R.G., and Cole, S.P.C. (2005). Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense. Toxicol. Appl. Pharmacol. 204, 216–237. Madan, B., Ke, Z., Harmston, N., Ho, S.Y., Frois, A.O., Alam, J., Jeyaraj, D.A., Pendharkar, V., Ghosh, K., Virshup, I.H., et al. (2016). Wnt addiction of genetically defined cancers reversed by PORCN inhibition. Oncogene 35, 2197–2207. Ng, M., Tan, D.S.P., Subbiah, V., Weekes, C.D., Teneggi, V., Diermayr, V., Ethirajulu, K., Yeo, P., Chen, D., Blanchard, S., et al. (2017). First-in-human phase 1 study of ETC-159 an oral PORCN inhbitor in patients with advanced solid tumours. J. Clin. Oncol. 35, 2584. Peng, K.C., Cluzeaud, F., Bens, M., Duong Van Huyen, J.P., Wioland, M.A., Lacave, R., and Vandewalle, A. (1999). Tissue and cell distribution of the multidrug resistance-associated protein (MRP) in mouse intestine and kidney. J. Histochem. Cytochem. 47, 757–768. Roulis, M., and Flavell, R.A. (2016). Fibroblasts and myofibroblasts of the intestinal lamina propria in physiology and disease. Differentiation 92, 116–131. San Roman, A.K., Jayewickreme, C.D., Murtaugh, L.C., and Shivdasani, R.A. (2014). Wnt secretion from epithelial cells and subepithelial myofibroblasts is not required in the mouse intestinal stem cell niche in vivo. Stem Cell Reports 2, 127–134. ten Hove, T., Drillenburg, P., Wijnholds, J., Te Velde, A.A., and van Deventer, S.J.H. (2002). Differential susceptibility of multidrug resistance protein-1 deficient mice to DSS and TNBS-induced colitis. Dig. Dis. Sci. 47, 2056–2063.
Previews The Myofibroblasts’ War on Drugs Jens Puschhof1,2 and Hans Clevers1,2,3,* 1Hubrecht
Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Uppsalalaan 8, 3584 CT Utrecht, the Netherlands Institute, Utrecht, the Netherlands 3Princess Ma ´ xima Centre, 3584 CT Utrecht, the Netherlands *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2018.09.008 2Oncode
The intestinal lining faces a constant assault from harmful gut contents. A study by Chee and colleagues in this issue of Developmental Cell describes how a population of myofibroblasts resists these threats and boosts regeneration of the intestinal epithelium. The human intestinal tract is constantly exposed to a plethora of potentially dangerous substances, ranging from poisonous compounds in food to bacterial metabolites and orally administered drugs. The epithelium lining the intestine is the first barrier against these compounds, and it copes with the incurred damage by active expulsion of small molecules and by renewing itself every 5 days. This renewal is driven by stem cells residing in intestinal crypts and supported by Wnt-producing epithelial Paneth cells. Another crucial source of Wnt niche signals are myofibroblasts located in the mesenchyme beneath the epithelium (Roulis and Flavell, 2016). Although these subepithelial myofibroblasts are exposed to similar threats from damaging luminal contents, they do not regenerate as quickly as the epithelium. To date, myofibroblasts’ mechanisms for coping with these insults have not been elucidated. A study in this issue of Developmental Cell (Chee et al., 2018) offers a compelling explanation for how the intestinal stroma resists hazardous intestinal contents. The study finds that a subpopulation of the myofibroblasts is intrinsically resistant to several xenobiotics and avoids excessive damage due to the expression of specific drug efflux pumps (Figure 1). The authors start by investigating a surprising discrepancy: shutting down Wnt ligand secretion through PORCN inhibition or genetic knockout in purely epithelial ‘‘mini-guts’’ (organoids) leads to complete disruption of the stem cell niche. In striking contrast, mice treated with comparable doses of PORCN inhibitors display negligible gut toxicity (Madan et al., 2016), and an early-stage clinical trial corroborates these findings in hu-
mans (Ng et al., 2017). The authors propose that the explanation for this is found in the myofibroblasts, as the sensitivity of epithelial organoids to PORCN inhibition was reduced when they were co-cultured with myofibroblasts. Previous reports had indeed found that non-epithelial Wnt sources stabilize the stem cell niche upon epithelial Porcn knockout (San Roman et al., 2014). Chee et al. (2018) found that co-culture with other Wnt-producing cells was not able to rescue PORCN inhibition-induced toxicity in intestinal organoids, hinting at a myofibroblast-specific resistance to PORCN inhibition. This observation prompted the authors to ask whether these myofibroblasts might also show an insensitivity to other xenobiotics. To answer this question and to investigate resistance to tamoxifen-induced recombination, the authors employed a Rosa26CreERT2;mT/mG mouse model. In this mouse model, all cells express membrane targeted tandem-dimer tomato (mT), which gets switched to membrane targeted green fluorescent protein (mG) upon tamoxifen-induced Cre activation, making it a useful tool to study global Cre activity. Consistent with the authors’ hypothesis, a subpopulation of subepithelial myofibroblasts did not switch color in response to tamoxifen, regardless of timings and route of administration, indicating that tamoxifen was not able to act in this population. The tamoxifen-resistant myofibroblasts were identified through expression of a-SMA and, in some cases, PDGFRa. In contrast, other tissues such as the liver showed recombination of the mT/mG allele in all cells and thus a complete tamoxifen response. Chee et al. (2018) go on to show that the tamoxifen-resistant population of myofibroblasts is also insensitive to the
anthracycline chemotherapeutic doxorubicin, highlighting its resistance to multiple xenobiotic agents. To determine whether the resistance to xenobiotics observed in this population of myofibroblasts was due to drug export from the cells, the authors used RNA sequencing to profile multidrug resistance transporter (MDR) expression in myofibroblasts. Intriguingly, Chee et al. (2018) found a subset of drug transporters including the ATP-binding cassette (ABC) transporters Abcc1 and Abcb1b overexpressed in myofibroblasts compared to epithelial crypts. In a set of elegant experiments, the authors identify ABCC1 as the dominant transporter relevant to myofibroblast resistance to PORCN inhibition. First, overexpression of ABCC1 in HEK293 cells increased expulsion of fluorescently labeled PORCN inhibitors. Second, specific inhibition of ABCC1 led to increased retention of PORCN inhibitor in myofibroblasts. Inhibition of ABCB1 had a smaller effect. These results are in line with experiments showing that while the intestines of Abcc1 KO and Abcb1 KO mice treated with PORCN inhibitors both displayed decreased expression of Wnt target genes as compared to WT controls, the difference was most notable in the Abcc1 KO intestines. Finally, ABCC1 inhibition also supported tamoxifen-induced recombination in the previously resistant myofibroblast population in vivo. Looking beyond the small intestine, the authors observe tamoxifen-resistant cell populations in organs such as the large intestine, lung, stomach, kidneys, and pancreas, but not in other tissues like liver and skeletal muscle. A more detailed characterization of these cell types will be essential to assessing their impact on
Developmental Cell 46, September 24, 2018 ª 2018 Elsevier Inc. 669
Developmental Cell
Previews
As PORCN inhibitors are currently being tested in clinical trials in patients with several types of cancers and ABC transporter inhibitors are being evaluated in combination with other anti-cancer therapies, this study has further important implications: inhibition of drug pumps in myofibroblasts may severely potentiate the gastrointestinal toxicity of compounds. Careful consideration of the differential resistance mechanisms of cancer cells and the intestinal stem cell niche may however yield viable strategies to selectively boost efficacy of anti-cancer drugs.
REFERENCES
Figure 1. Myofibroblasts Provide a Xenobiotic-Resistant Stem Cell Niche through ABCC1 Transporter Expression Xenobiotics from the intestinal lumen harm the epithelial cells in the crypt but are actively expelled from subepithelial myofibroblasts through ABCC1 transporters. Epithelial Paneth cells stop secreting Wnt ligands in response to some xenobiotics, which is rescued by myofibroblast-derived Wnt ligands. Image adapted from Servier Medical Art (https://smart.servier.com/).
lineage tracing studies in these organs and to forming an understanding of their role in response to damaging agents. The results presented by Chee et al. (2018) require that the field use caution when interpreting the results of previous reports using chemically induced activation and recombination of fibroblasts. In a previous study, a tamoxifen-induced simultaneous epithelial and subepithelial knockout of Porcn did not induce major gut disruption (San Roman et al., 2014). The new results suggest that these experiments did not yield a phenotype because of a tamoxifen-resistant population of myofibroblasts that did not lose Porcn expression and was thus able to continue providing the stem cell niche. For the large intestine, a recent study (Degirmenci et al., 2018) describes an essential Wnt ligand producing mesenchymal population marked by GLI1 and PDGFRa. This population was identified through tamoxifen-induced recombination and therefore appears to be sensitive to this xenobiotic. These findings highlight the need for a more detailed characterization of the resistant myofibroblast population in the small intestine, contrasting it to other xenobiotic-sensitive mesenchymal cell populations. The findings presented provide a new angle on the response of the intestinal stem cell niche under xenobiotic stress.
The importance of Abcc1 expression for myofibroblast resistance to several compounds is convincingly demonstrated. While the resistance to tamoxifen and PORCN inhibitors is highly relevant to genetic studies and translational efforts, more nuanced investigations will be necessary to elucidate the cell-type-specific role of individual ABC transporters in response to natural compounds. In particular, considering bacterial metabolites could yield important translational insights, as an Abcc1 / model showed increased susceptibility toward developing inflammatory bowel disease (IBD) (ten Hove et al., 2002). Since the epithelium is even more directly exposed to luminal compounds, it is of high interest to understand why only myofibroblasts would express the described subset of ABC transporters. The authors have also identified a set of ABC transporters enriched in epithelial cells versus myofibroblasts, which adds more detail to previous reports (Leslie et al., 2005; Peng et al., 1999). Further studies on the differential response to hazardous compounds between epithelium and mesenchyme may shed light on specialized resistance mechanisms in both compartments and provide insights as to how niche signals beyond Wnt ligands are provided under xenobiotic stress conditions.
670 Developmental Cell 46, September 24, 2018
Chee, Y.C., Pahnke, J., Bunte, R., Adsool, V.A., Madan, B., and Virshup, D.M. (2018). Intrinsic xenobiotic resistance of the intestinal stem cell niche. Dev. Cell 46, this issue, 681–695. Degirmenci, B., Valenta, T., Dimitrieva, S., Hausmann, G., and Basler, K. (2018). GLI1-expressing mesenchymal cells form the essential Wntsecreting niche for colon stem cells. Nature 558, 449–453. Leslie, E.M., Deeley, R.G., and Cole, S.P.C. (2005). Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense. Toxicol. Appl. Pharmacol. 204, 216–237. Madan, B., Ke, Z., Harmston, N., Ho, S.Y., Frois, A.O., Alam, J., Jeyaraj, D.A., Pendharkar, V., Ghosh, K., Virshup, I.H., et al. (2016). Wnt addiction of genetically defined cancers reversed by PORCN inhibition. Oncogene 35, 2197–2207. Ng, M., Tan, D.S.P., Subbiah, V., Weekes, C.D., Teneggi, V., Diermayr, V., Ethirajulu, K., Yeo, P., Chen, D., Blanchard, S., et al. (2017). First-in-human phase 1 study of ETC-159 an oral PORCN inhbitor in patients with advanced solid tumours. J. Clin. Oncol. 35, 2584. Peng, K.C., Cluzeaud, F., Bens, M., Duong Van Huyen, J.P., Wioland, M.A., Lacave, R., and Vandewalle, A. (1999). Tissue and cell distribution of the multidrug resistance-associated protein (MRP) in mouse intestine and kidney. J. Histochem. Cytochem. 47, 757–768. Roulis, M., and Flavell, R.A. (2016). Fibroblasts and myofibroblasts of the intestinal lamina propria in physiology and disease. Differentiation 92, 116–131. San Roman, A.K., Jayewickreme, C.D., Murtaugh, L.C., and Shivdasani, R.A. (2014). Wnt secretion from epithelial cells and subepithelial myofibroblasts is not required in the mouse intestinal stem cell niche in vivo. Stem Cell Reports 2, 127–134. ten Hove, T., Drillenburg, P., Wijnholds, J., Te Velde, A.A., and van Deventer, S.J.H. (2002). Differential susceptibility of multidrug resistance protein-1 deficient mice to DSS and TNBS-induced colitis. Dig. Dis. Sci. 47, 2056–2063.