- Email: [email protected]
Role Reversal: A Pro-metastatic Function of E-Cadherin
Developmental Cell
Previews WASP and involves Nck, WIP, and Grb2. J. Biol. Chem. 277, 37771–37776. €nisch, J., Ehinger, J., Ladwein, M., Rohde, M., Ha Derivery, E., Bosse, T., Steffen, A., Bumann, D., Misselwitz, B., Hardt, W.D., et al. (2010). Molecular dissection of Salmonella-induced membrane ruffling versus invasion. Cell. Microbiol. 12, 84–98. Juin, A., Spence, H.J., Martin, K.J., McGhee, E., Neilson, M., Cutiongco, M.F.A., Gadegaard, N., Mackay, G., Fort, L., Lilla, S., et al. (2019). NWASP control of LPAR1 trafficking establishes
response to self-generated LPA gradients to promote pancreatic cancer cell metastasis. Dev. Cell 51, this issue, 431–445. Li, H., and Brakebusch, C. (2019). Senescence regulation by nuclear N-WASP: a role in cancer? Oncoscience 6, 354–356. Molinie, N., and Gautreau, A. (2018). The Arp2/3 regulatory system and its deregulation in cancer. Physiol. Rev. 98, 215–238. Muinonen-Martin, A.J., Susanto, O., Zhang, Q., Smethurst, E., Faller, W.J., Veltman, D.M., Kalna,
G., Lindsay, C., Bennett, D.C., Sansom, O.J., et al. (2014). Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal. PLoS Biol. 12, e1001966. Rodal, A.A., Motola-Barnes, R.N., and Littleton, J.T. (2008). Nervous wreck and Cdc42 cooperate to regulate endocytic actin assembly during synaptic growth. J. Neurosci. 28, 8316–8325. Sheng, X., Yung, Y.C., Chen, A., and Chun, J. (2015). Lysophosphatidic acid signalling in development. Development 142, 1390–1395.
Role Reversal: A Pro-metastatic Function of E-Cadherin Minhong Shen1 and Yibin Kang1,* 1Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2019.10.028
Carcinoma cells often acquire mobility and invasiveness by undergoing epithelial-to-mesenchymal transition, and yet most metastatic lesions remain epithelial in nature. Recently, in Nature, Padmanaban et al. (2019) demonstrated that the quintessential epithelial marker E-cadherin promotes metastasis of invasive ductal breast carcinoma by enhancing the survival of tumor cells. Most of cancer mortality is caused by the metastatic spread of tumors to other organs. Previous studies indicated that tumor cells gain mobility and invasiveness by losing epithelial features while gaining mesenchymal characteristics, through a process called epithelial-to-mesenchymal transition (EMT) (Yang et al., 2004). On the other hand, mesenchymal-to-epithelial transition (MET), which restores the epithelial properties of cancer cells, has also been reported to be essential for metastatic colonization (Tsai et al., 2012). Therefore, the epithelial/mesenchymal status of cancer cells needs to be dynamically regulated to allow successful formation of metastasis. While tremendous progress has been made in understanding the gene regulatory network of EMT and its biological consequence (Lu and Kang, 2019), how MET and the epithelial features of cancer cells contribute to metastasis remains relatively unknown. The transmembrane protein E-cadherin is one of the crucial molecules for the formation of adhesive intercellular junctions between epithelial cells and for the estab-
lishment of cellular polarity and is therefore considered a hallmark of epithelial status. Frequent inactivating mutations or epigenetic silencing of E-cadherin have been observed in multiple cancers, such as diffuse gastric cancers and invasive lobular carcinoma (ILC) of the breast (Berx et al., 1998). Functionally, E-cadherin restricts the cell migration in vitro, and its levels are inversely correlated with invasion capacity in vivo (Canel et al., 2013; Onder et al., 2008), suggesting a metastasis suppressor role for E-cadherin. However, invasive ductal carcinomas (IDCs) often retain E-cadherin expression in primary tumors and metastases (Li et al., 2003), suggesting possible requirement of E-cadherin-mediated functions for metastasis of IDC. To explore potentially differential roles of E-cadherin in metastasis, in a recent study in Nature Padmanaban et al. used elegant mouse models and in vitro cellular assays to dissect the function of E-cadherin in IDC (Padmanaban et al., 2019). The researchers first used the MMTVPyMT breast tumor model, which repre-
sents luminal IDC that expresses E-cadherin during tumor growth, invasion, dissemination, and metastatic colonization. Genetic engineering in this model allowed E-cadherin to be conditionally depleted by Cre recombinase, and the resulting E-cadherin cells were marked by GFP in contrast to the mTomato-labeled E-cadherin+ cells. Taking advantage of this system, Padmanaban et al. found that E-cadherin loss increases invasion and dissemination in vitro, which is consistent with previous studies showing that E-cadherin suppresses cell invasion and behaves as a metastasis suppressor (Canel et al., 2013; Onder et al., 2008). Surprisingly, instead of promoting distant metastasis, cells with E-cadherin loss generated significantly less lung metastases (Figure 1). Similar observations were also made in multiple mouse and PDX models of IDCs to rule out possible complications arising from the oncogenic driver, donor mice, immune microenvironment, and cell non-autonomous effects. To understand the functional importance of E-cadherin in the multi-step
Developmental Cell 51, November 18, 2019 ª 2019 Published by Elsevier Inc. 417
Developmental Cell
Previews
Figure 1. E-Cadherin Promotes Breast Cancer Metastasis by Conferring Survival Advantage Breast IDC tumors with E-cadherin loss (bottom panel) are associated with TGF-b signaling activation, which in turn increases oxidative stress and activates ROS. The elevated ROS induces cell apoptosis and subsequently reduces CTC number as well as distant metastasis. IDC, invasive ductal carcinoma; E-cadherin+, E-cadherin wild-type cells; E-cadherin-, E-cadherin-knockout cells; CTCs, circulating tumor cells; ROS, reactive oxygen species.
process of metastasis, Padmanaban et al. utilized several in vitro and in vivo model systems. E-cadherin depletion reduced colony formation and tumor growth in 3D matrigel culture as well as the number of circulating tumor cells in vivo. Seeding of micrometastasis from intravenously injected tumors cells was also dramatically reduced. These results point to a critical role of E-cadherin in supporting the survival of cancer cells during the growth of the primary, dissemination through circulation, and initial seeding of metastasis. A previous study indicated that downregulation of E-cadherin and loss of epithelial features during EMT circumvents breast cancer cell dormancy and is required to initiate metastatic outgrowth (Wendt et al., 2011). However, no differ-
ences in the dormancy marker NR2F1 were observed between E-cadherin wild-type and knockout cells. Moreover, no significant shift of EMT transcriptome or markers was observed upon E-cadherin depletion, suggesting that the functional impact of E-cadherin in metastasis is not likely to be just a side product of its association with the epithelial status. To investigate the underlying mechanism of the pro-metastasis role of E-cadherin, RNA-seq analysis was performed and indicated a strong positive association of E-cadherin loss with apoptotic and stress-related pathways. Indeed, E-cadherin loss dramatically increased apoptosis in vitro and in vivo, suggesting that E-cadherin sustains tumor cell survival under stressful situations to promote
418 Developmental Cell 51, November 18, 2019
metastasis. Through mechanisms that remain uncharacterized, E-cadherin loss activates TGF-b signaling, as well as TNFa and p53 pathways, which in turn elevates oxidative stress and reactive oxygen species (ROS) to suppress metastasis (Figure 1). Pharmacological inhibition of TGF-bR1 and ROS rescues the metastatic ability of tumor cells after E-cadherin loss. Collectively, the study in Nature established a functional role of E-cadherin in promoting metastasis of IDC through a pro-survival signaling axis (Figure 1). Loss or gain of epithelial fate through EMT/MET at different stages of the metastatic cascade was previously thought to be critical for cancer metastasis. However, accumulating evidence points to a partial EMT status and high degree of plasticity as more conducive to efficient development of metastasis (reviewed in Lu and Kang, 2019). As an epithelial marker, E-cadherin depletion did not abolish the epithelial features in this study, suggesting that E-cadherin is not absolutely required to lock tumor cells in epithelial status in IDC. Rather, a hybrid state might confer collective migratory ability to tumor cells while allowing them to survive during transit and colonization in distant organs. It should also be noted that the functional requirement for E-cadherin in metastasis is likely to be highly context dependent. The pro-metastatic function of E-cadherin and its related signaling mechanism need to be further explored in cancer types that retain or even elevate E-cadherin expression, such as inflammatory breast carcinoma, ovarian cancer, and some subset of brain tumors (Rodriguez et al., 2012). In diffuse gastric cancers and ILC of the breast where E-cadherin is frequently mutated or silenced, its tumor- and metastasis-suppressive role is likely to be more prevalent, as has been validated in some previous functional studies (Onder et al., 2008). Key questions that remain to be resolved are why and how does E-cadherin play such differential roles in a context-dependent manner? Could this be the result of different sensitivity of tumor cells to TGF-b-mediated ROS generation and subsequent induction of apoptosis? It is also possible that other regulators of EMT, such as EMT transcription factors and the miR-200 family of miRNAs, synergize with or counteract
Developmental Cell
Previews against E-cadeherin in metastasis. As such, the exact status of the cancer cells in the epithelial/mesenchymal spectrum may determine the functional readout of E-cadherin in metastasis. Further studies to explore these questions will help define possible windows for E-cadherin-targeting therapeutic intervention. REFERENCES Berx, G., Becker, K.F., Ho¨fler, H., and van Roy, F. (1998). Mutations of the human E-cadherin (CDH1) gene. Hum. Mutat. 12, 226–237. Canel, M., Serrels, A., Frame, M.C., and Brunton, V.G. (2013). E-cadherin-integrin crosstalk in cancer invasion and metastasis. J. Cell Sci. 126, 393–401.
Li, C.I., Anderson, B.O., Daling, J.R., and Moe, R.E. (2003). Trends in incidence rates of invasive lobular and ductal breast carcinoma. JAMA 289, 1421–1424. Lu, W., and Kang, Y. (2019). Epithelial-mesenchymal plasticity in cancer progression and metastasis. Dev. Cell 49, 361–374. Onder, T.T., Gupta, P.B., Mani, S.A., Yang, J., Lander, E.S., and Weinberg, R.A. (2008). Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res. 68, 3645–3654. Padmanaban, V., Krol, I., Suhail, Y., Szczerba, B.M., Aceto, N., Bader, J.S., and Ewald, A.J. (2019). E-cadherin is required for metastasis in multiple models of breast cancer. Nature 573, 439–444. Rodriguez, F.J., Lewis-Tuffin, L.J., and Anastasiadis, P.Z. (2012). E-cadherin’s dark side:
possible role in tumor progression. Biochim. Biophys. Acta 1826, 23–31. Tsai, J.H., Donaher, J.L., Murphy, D.A., Chau, S., and Yang, J. (2012). Spatiotemporal regulation of epithelial-mesenchymal transition is essential for squamous cell carcinoma metastasis. Cancer Cell 22, 725–736. Wendt, M.K., Taylor, M.A., Schiemann, B.J., and Schiemann, W.P. (2011). Down-regulation of epithelial cadherin is required to initiate metastatic outgrowth of breast cancer. Mol. Biol. Cell 22, 2423–2435. Yang, J., Mani, S.A., Donaher, J.L., Ramaswamy, S., Itzykson, R.A., Come, C., Savagner, P., Gitelman, I., Richardson, A., and Weinberg, R.A. (2004). Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117, 927–939.
Developmental Cell 51, November 18, 2019 419
Previews WASP and involves Nck, WIP, and Grb2. J. Biol. Chem. 277, 37771–37776. €nisch, J., Ehinger, J., Ladwein, M., Rohde, M., Ha Derivery, E., Bosse, T., Steffen, A., Bumann, D., Misselwitz, B., Hardt, W.D., et al. (2010). Molecular dissection of Salmonella-induced membrane ruffling versus invasion. Cell. Microbiol. 12, 84–98. Juin, A., Spence, H.J., Martin, K.J., McGhee, E., Neilson, M., Cutiongco, M.F.A., Gadegaard, N., Mackay, G., Fort, L., Lilla, S., et al. (2019). NWASP control of LPAR1 trafficking establishes
response to self-generated LPA gradients to promote pancreatic cancer cell metastasis. Dev. Cell 51, this issue, 431–445. Li, H., and Brakebusch, C. (2019). Senescence regulation by nuclear N-WASP: a role in cancer? Oncoscience 6, 354–356. Molinie, N., and Gautreau, A. (2018). The Arp2/3 regulatory system and its deregulation in cancer. Physiol. Rev. 98, 215–238. Muinonen-Martin, A.J., Susanto, O., Zhang, Q., Smethurst, E., Faller, W.J., Veltman, D.M., Kalna,
G., Lindsay, C., Bennett, D.C., Sansom, O.J., et al. (2014). Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal. PLoS Biol. 12, e1001966. Rodal, A.A., Motola-Barnes, R.N., and Littleton, J.T. (2008). Nervous wreck and Cdc42 cooperate to regulate endocytic actin assembly during synaptic growth. J. Neurosci. 28, 8316–8325. Sheng, X., Yung, Y.C., Chen, A., and Chun, J. (2015). Lysophosphatidic acid signalling in development. Development 142, 1390–1395.
Role Reversal: A Pro-metastatic Function of E-Cadherin Minhong Shen1 and Yibin Kang1,* 1Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2019.10.028
Carcinoma cells often acquire mobility and invasiveness by undergoing epithelial-to-mesenchymal transition, and yet most metastatic lesions remain epithelial in nature. Recently, in Nature, Padmanaban et al. (2019) demonstrated that the quintessential epithelial marker E-cadherin promotes metastasis of invasive ductal breast carcinoma by enhancing the survival of tumor cells. Most of cancer mortality is caused by the metastatic spread of tumors to other organs. Previous studies indicated that tumor cells gain mobility and invasiveness by losing epithelial features while gaining mesenchymal characteristics, through a process called epithelial-to-mesenchymal transition (EMT) (Yang et al., 2004). On the other hand, mesenchymal-to-epithelial transition (MET), which restores the epithelial properties of cancer cells, has also been reported to be essential for metastatic colonization (Tsai et al., 2012). Therefore, the epithelial/mesenchymal status of cancer cells needs to be dynamically regulated to allow successful formation of metastasis. While tremendous progress has been made in understanding the gene regulatory network of EMT and its biological consequence (Lu and Kang, 2019), how MET and the epithelial features of cancer cells contribute to metastasis remains relatively unknown. The transmembrane protein E-cadherin is one of the crucial molecules for the formation of adhesive intercellular junctions between epithelial cells and for the estab-
lishment of cellular polarity and is therefore considered a hallmark of epithelial status. Frequent inactivating mutations or epigenetic silencing of E-cadherin have been observed in multiple cancers, such as diffuse gastric cancers and invasive lobular carcinoma (ILC) of the breast (Berx et al., 1998). Functionally, E-cadherin restricts the cell migration in vitro, and its levels are inversely correlated with invasion capacity in vivo (Canel et al., 2013; Onder et al., 2008), suggesting a metastasis suppressor role for E-cadherin. However, invasive ductal carcinomas (IDCs) often retain E-cadherin expression in primary tumors and metastases (Li et al., 2003), suggesting possible requirement of E-cadherin-mediated functions for metastasis of IDC. To explore potentially differential roles of E-cadherin in metastasis, in a recent study in Nature Padmanaban et al. used elegant mouse models and in vitro cellular assays to dissect the function of E-cadherin in IDC (Padmanaban et al., 2019). The researchers first used the MMTVPyMT breast tumor model, which repre-
sents luminal IDC that expresses E-cadherin during tumor growth, invasion, dissemination, and metastatic colonization. Genetic engineering in this model allowed E-cadherin to be conditionally depleted by Cre recombinase, and the resulting E-cadherin cells were marked by GFP in contrast to the mTomato-labeled E-cadherin+ cells. Taking advantage of this system, Padmanaban et al. found that E-cadherin loss increases invasion and dissemination in vitro, which is consistent with previous studies showing that E-cadherin suppresses cell invasion and behaves as a metastasis suppressor (Canel et al., 2013; Onder et al., 2008). Surprisingly, instead of promoting distant metastasis, cells with E-cadherin loss generated significantly less lung metastases (Figure 1). Similar observations were also made in multiple mouse and PDX models of IDCs to rule out possible complications arising from the oncogenic driver, donor mice, immune microenvironment, and cell non-autonomous effects. To understand the functional importance of E-cadherin in the multi-step
Developmental Cell 51, November 18, 2019 ª 2019 Published by Elsevier Inc. 417
Developmental Cell
Previews
Figure 1. E-Cadherin Promotes Breast Cancer Metastasis by Conferring Survival Advantage Breast IDC tumors with E-cadherin loss (bottom panel) are associated with TGF-b signaling activation, which in turn increases oxidative stress and activates ROS. The elevated ROS induces cell apoptosis and subsequently reduces CTC number as well as distant metastasis. IDC, invasive ductal carcinoma; E-cadherin+, E-cadherin wild-type cells; E-cadherin-, E-cadherin-knockout cells; CTCs, circulating tumor cells; ROS, reactive oxygen species.
process of metastasis, Padmanaban et al. utilized several in vitro and in vivo model systems. E-cadherin depletion reduced colony formation and tumor growth in 3D matrigel culture as well as the number of circulating tumor cells in vivo. Seeding of micrometastasis from intravenously injected tumors cells was also dramatically reduced. These results point to a critical role of E-cadherin in supporting the survival of cancer cells during the growth of the primary, dissemination through circulation, and initial seeding of metastasis. A previous study indicated that downregulation of E-cadherin and loss of epithelial features during EMT circumvents breast cancer cell dormancy and is required to initiate metastatic outgrowth (Wendt et al., 2011). However, no differ-
ences in the dormancy marker NR2F1 were observed between E-cadherin wild-type and knockout cells. Moreover, no significant shift of EMT transcriptome or markers was observed upon E-cadherin depletion, suggesting that the functional impact of E-cadherin in metastasis is not likely to be just a side product of its association with the epithelial status. To investigate the underlying mechanism of the pro-metastasis role of E-cadherin, RNA-seq analysis was performed and indicated a strong positive association of E-cadherin loss with apoptotic and stress-related pathways. Indeed, E-cadherin loss dramatically increased apoptosis in vitro and in vivo, suggesting that E-cadherin sustains tumor cell survival under stressful situations to promote
418 Developmental Cell 51, November 18, 2019
metastasis. Through mechanisms that remain uncharacterized, E-cadherin loss activates TGF-b signaling, as well as TNFa and p53 pathways, which in turn elevates oxidative stress and reactive oxygen species (ROS) to suppress metastasis (Figure 1). Pharmacological inhibition of TGF-bR1 and ROS rescues the metastatic ability of tumor cells after E-cadherin loss. Collectively, the study in Nature established a functional role of E-cadherin in promoting metastasis of IDC through a pro-survival signaling axis (Figure 1). Loss or gain of epithelial fate through EMT/MET at different stages of the metastatic cascade was previously thought to be critical for cancer metastasis. However, accumulating evidence points to a partial EMT status and high degree of plasticity as more conducive to efficient development of metastasis (reviewed in Lu and Kang, 2019). As an epithelial marker, E-cadherin depletion did not abolish the epithelial features in this study, suggesting that E-cadherin is not absolutely required to lock tumor cells in epithelial status in IDC. Rather, a hybrid state might confer collective migratory ability to tumor cells while allowing them to survive during transit and colonization in distant organs. It should also be noted that the functional requirement for E-cadherin in metastasis is likely to be highly context dependent. The pro-metastatic function of E-cadherin and its related signaling mechanism need to be further explored in cancer types that retain or even elevate E-cadherin expression, such as inflammatory breast carcinoma, ovarian cancer, and some subset of brain tumors (Rodriguez et al., 2012). In diffuse gastric cancers and ILC of the breast where E-cadherin is frequently mutated or silenced, its tumor- and metastasis-suppressive role is likely to be more prevalent, as has been validated in some previous functional studies (Onder et al., 2008). Key questions that remain to be resolved are why and how does E-cadherin play such differential roles in a context-dependent manner? Could this be the result of different sensitivity of tumor cells to TGF-b-mediated ROS generation and subsequent induction of apoptosis? It is also possible that other regulators of EMT, such as EMT transcription factors and the miR-200 family of miRNAs, synergize with or counteract
Developmental Cell
Previews against E-cadeherin in metastasis. As such, the exact status of the cancer cells in the epithelial/mesenchymal spectrum may determine the functional readout of E-cadherin in metastasis. Further studies to explore these questions will help define possible windows for E-cadherin-targeting therapeutic intervention. REFERENCES Berx, G., Becker, K.F., Ho¨fler, H., and van Roy, F. (1998). Mutations of the human E-cadherin (CDH1) gene. Hum. Mutat. 12, 226–237. Canel, M., Serrels, A., Frame, M.C., and Brunton, V.G. (2013). E-cadherin-integrin crosstalk in cancer invasion and metastasis. J. Cell Sci. 126, 393–401.
Li, C.I., Anderson, B.O., Daling, J.R., and Moe, R.E. (2003). Trends in incidence rates of invasive lobular and ductal breast carcinoma. JAMA 289, 1421–1424. Lu, W., and Kang, Y. (2019). Epithelial-mesenchymal plasticity in cancer progression and metastasis. Dev. Cell 49, 361–374. Onder, T.T., Gupta, P.B., Mani, S.A., Yang, J., Lander, E.S., and Weinberg, R.A. (2008). Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res. 68, 3645–3654. Padmanaban, V., Krol, I., Suhail, Y., Szczerba, B.M., Aceto, N., Bader, J.S., and Ewald, A.J. (2019). E-cadherin is required for metastasis in multiple models of breast cancer. Nature 573, 439–444. Rodriguez, F.J., Lewis-Tuffin, L.J., and Anastasiadis, P.Z. (2012). E-cadherin’s dark side:
possible role in tumor progression. Biochim. Biophys. Acta 1826, 23–31. Tsai, J.H., Donaher, J.L., Murphy, D.A., Chau, S., and Yang, J. (2012). Spatiotemporal regulation of epithelial-mesenchymal transition is essential for squamous cell carcinoma metastasis. Cancer Cell 22, 725–736. Wendt, M.K., Taylor, M.A., Schiemann, B.J., and Schiemann, W.P. (2011). Down-regulation of epithelial cadherin is required to initiate metastatic outgrowth of breast cancer. Mol. Biol. Cell 22, 2423–2435. Yang, J., Mani, S.A., Donaher, J.L., Ramaswamy, S., Itzykson, R.A., Come, C., Savagner, P., Gitelman, I., Richardson, A., and Weinberg, R.A. (2004). Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117, 927–939.
Developmental Cell 51, November 18, 2019 419