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Unsaturated Fatty Acids Maintain Cancer Cell Stemness
Cell Stem Cell
Previews Unsaturated Fatty Acids Maintain Cancer Cell Stemness Abir Mukherjee,1 Hilary A. Kenny,1 and Ernst Lengyel1,* 1Department of Obstetrics and Gynecology/Section of Gynecologic Oncology, The University of Chicago, Chicago, IL 60637, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2017.02.008
Investigation of the metabolic regulation of cancer stem cells is an emerging field that offers promising approaches for identifying and targeting recalcitrant stem cell populations. In this issue of Cell Stem Cell, Li et al. (2017) indicate that increased lipid desaturation is essential to stem-like characteristics in ovarian cancer cells. A small but significant percentage of all cancer cells are considered stem-like cells, based on their self-renewal and tumor-initiating properties. Cancer stem cells (CSCs) are resistant to standard chemotherapy and are therefore implicated in disease progression, recurrence, and adverse patient outcomes. Evidence now points to a CSC-driven hierarchal model of tumor growth, contributing to intra-tumoral heterogeneity and differential responses to chemotherapy. Hence, the need of the hour is to reproducibly identify and characterize CSC populations as well as understand the mechanisms that govern their differentiation and pro-metastatic function(s). Several studies have elucidated cellular signaling pathways important to the maintenance of cancer cell stemness; however, the study of metabolic pathways in the regulation of stemness is in its infancy. One of the few indications of CSC metabolic regulation was found in basallike breast cancer, where the transition of cells along the EMT continuum induces aerobic glycolysis and suppresses ROS production. Switching to glycolysis enhances the cells’ CSC characteristics and allows them to withstand hypoxia in the malignant environment (Dong et al., 2013). In this issue of Cell Stem Cell, Li et al. (2017) provide new insights into the interdependency of metabolism and stem cell characteristics by comparing the lipid content and composition of ALDH+ CD133+ ovarian CSCs with ALDH CD133 cancer cells. In an elegantly performed study, the authors show that ovarian CSCs and cancer cells grown as spheroids have a higher ratio of unsaturated to saturated fatty acids and that
this ratio is essential for the cells to retain stemness (Figure 1). To perform the study, Li et al. use single-cell stimulated Raman scattering (SRS) microscopy to compare the lipid content and the levels of unsaturation in the intracellular lipid pools of living single cells. By scanning single cells with a femtosecond laser and measuring Raman shifts pertaining to lipid C-H vibrations, the authors show that ovarian CSCs have significantly higher levels of lipid droplets and that these droplets contain a higher percentage of unsaturated lipids. They then go on to confirm these findings by using primary cancer cells isolated from ascites and grown as spheroids.
Given that spheroid culture increases cellular stem-like properties, their data suggest that increases in lipid unsaturation might be a general marker for CSCs in ovarian cancer. In order to determine whether lipid characteristics may be a more generalizable marker for CSC populations, it will be necessary to test multiple CSC populations from different cancers, each isolated using separate cell surface markers. However, the data presented herein strongly suggest that fatty acid desaturation is a metabolic marker of ovarian CSC populations. Fatty acid desaturases are a class of enzymes that create double bonds at
virtual energy state O
9
OH
10
saturated lipids SCD-1 O 9 10
vibrational energy state
OH
unsaturated lipids NF-κB
SCD -1 ALDH1A1
stemness
Figure 1. Higher Unsaturated Lipid Profile of Ovarian Cancer Stem Cells Attributed to Increased SCD-1 Expression Changes in lipid profile are detected using stimulated Raman scattering (SRS) microscopy. Ovarian cancer cells grown as spheroids have a higher lipid content, especially unsaturated lipids, which scatter monochromatic light differently (left panel) as depicted in the energy level diagram. The authors use this method and show that ovarian cancer stem cells (CSCs) (ALDH+/CD133+) have increased lipid droplets. Growing ovarian CSCs and cell lines as spheroids increases their unsaturated lipid content, which activates NFkB activity, thereby increasing SCD-1 and ALDH1A1 expression. These proteins subsequently increase and maintain stemness of cancer cells, thus inducing a feedforward loop where unsaturated fatty acids increase SCD-1 via NFkB, leading to enhanced unsaturated fatty acid production.
Cell Stem Cell 20, March 2, 2017 ª 2017 Elsevier Inc. 291
Cell Stem Cell
Previews specific locations in long chain fatty acids. For example palmitic acid (16:0) becomes palmitoleic acid (16:1) with a double bond at the ninth carbon position. StearoylCoA desaturase-1 (SCD-1), the most abundant desaturase, is expressed in lipogenic tissues and catalyzes the formation of double bonds at the ninth carbon atom of saturated fatty acids, leading to monounsaturated fatty acids (MUFA). Using chemical inhibitors and molecular approaches (shRNA), the authors identify SCD-1 as the enzyme responsible for the increased desaturation in stem cells. They show that blocking SCD-1 significantly reduces the stem cell population, as evidenced by inhibition of several stem cell markers, including aldehyde dehydrogenase activity, in vitro. Blocking SCD-1 in ovarian cancer spheroids before injecting them subcutaneously into mice reduces the cells’ capacity to initiate tumors and inhibits their proliferation, resulting in very small tumors. Previous reports have shown that SCD-1 is regulated by multiple oncogenic signals and that its expression correlates with cell-cycle progression and proliferation (Demoulin et al., 2004), as well as reduced apoptosis and lipid-mediated cytotoxicity (Scaglia and Igal, 2005). In addition, there is strong evidence in the literature that abnormal fatty acid composition, especially an increased MUFA to saturated fatty acid ratio, is present in several cancer types. High levels of SCD-1 and MUFA are associated with an adverse patient prognosis, especially in aggressive breast cancer (Chaje`s et al., 1999). Li et al. complements these studies by demonstrating that SCD-1 and MUFA play critical roles in the maintenance of the CSC phenotype. Moreover, the authors reinforce the view that lipids are not just a static energy depot, but rather that lipids are important modulators of the cancer phenotype. This study pro-
292 Cell Stem Cell 20, March 2, 2017
vides formative evidence that a specific lipid composition and an abundance of lipid droplets promote cancer stemness, corroborating the idea that metabolism affects every aspect of the cancer phenotype. Finally, using an unbiased approach, the authors identify several stem cellrelated pathways affected by SCD-1 inhibition. They demonstrate that NFkB signaling regulates SCD-1, lipid unsaturation, and, in consequence, stemness. It is particularly interesting that increased lipid unsaturation and active NFkB signaling upregulate ALDH1A1 mRNA levels and that NFkB inhibition abrogates this process. These findings place NFkB at the center of unsaturated lipid-mediated regulation of stemness in ovarian cancer cells. The findings also corroborate previous reports that ovarian cancer cells take up lipids from their adipocyte-rich tumor microenvironment through an FABP4dependent shuttling mechanism (Nieman et al., 2011) that is known to affect the NFkB pathway (Makowski et al., 2005). In summary, this paper is an important contribution to the CSC literature, as it demonstrates that SCD-1 is critical to maintaining the ovarian CSC phenotype by globally affecting lipogenesis. Chemotherapy is effective for non-stem cells, so SCD-1 inhibition seems to be a promising therapeutic option to specifically target stem cells and prolong patient survival. Several inhibitors of SCD-1 have been developed, mostly for diabetes treatment; however, testing has not gone beyond phase II clinical trials (Zhang et al., 2014). While the trials have not been formally published, deletion of the SCD-1 gene in a mouse model reportedly causes mechanism-based adverse effects such as narrow eye fissures, alopecia, loss of sebaceous gland function, and skin abnormalities (Miyazaki et al.,
2001), raising concerns that generalized blocking of SCD-1 may not be a clinically viable approach. However, given the convincing findings of this paper, and the urgent need for effective ovarian cancer treatments especially against stem cells, the identification of a SCD-1 inhibitor that can be clinically tolerated is clearly a worthwhile future pursuit. ACKNOWLEDGMENTS This work was supported by grants from the ovarian cancer research fund alliance (Ann and Sol Schreiber Mentored Investigator Award, awarded to A.M.) and National Cancer Institute/ National Institute of Health (R01CA169604-01A1 awarded to E.L.). We also thank Gail Isenberg (University of Chicago) for carefully editing this manuscript.
REFERENCES Chaje`s, V., Hulte´n, K., Van Kappel, A.L., Winkvist, A., Kaaks, R., Hallmans, G., Lenner, P., and Riboli, E. (1999). Int. J. Cancer 83, 585–590. Demoulin, J.B., Ericsson, J., Kallin, A., Rorsman, C., Ro¨nnstrand, L., and Heldin, C.H. (2004). J. Biol. Chem. 279, 35392–35402. Dong, C., Yuan, T., Wu, Y., Wang, Y., Fan, T.W., Miriyala, S., Lin, Y., Yao, J., Shi, J., Kang, T., et al. (2013). Cancer Cell 23, 316–331. Li, J., Condello, S., Thomes-Pepin, J., Ma, X., Xia, Y., Hurley, T.D., Matei, D., and Cheng, J.X. (2017). Cell Stem Cell 20, this issue, 303–314. Makowski, L., Brittingham, K.C., Reynolds, J.M., Suttles, J., and Hotamisligil, G.S. (2005). J. Biol. Chem. 280, 12888–12895. Miyazaki, M., Man, W.C., and Ntambi, J.M. (2001). J. Nutr. 131, 2260–2268. Nieman, K.M., Kenny, H.A., Penicka, C.V., Ladanyi, A., Buell-Gutbrod, R., Zillhardt, M.R., Romero, I.L., Carey, M.S., Mills, G.B., Hotamisligil, G.S., et al. (2011). Nat. Med. 17, 1498–1503. Scaglia, N., and Igal, R.A. (2005). J. Biol. Chem. 280, 25339–25349. Zhang, Z., Dales, N.A., and Winther, M.D. (2014). J. Med. Chem. 57, 5039–5056.
Previews Unsaturated Fatty Acids Maintain Cancer Cell Stemness Abir Mukherjee,1 Hilary A. Kenny,1 and Ernst Lengyel1,* 1Department of Obstetrics and Gynecology/Section of Gynecologic Oncology, The University of Chicago, Chicago, IL 60637, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2017.02.008
Investigation of the metabolic regulation of cancer stem cells is an emerging field that offers promising approaches for identifying and targeting recalcitrant stem cell populations. In this issue of Cell Stem Cell, Li et al. (2017) indicate that increased lipid desaturation is essential to stem-like characteristics in ovarian cancer cells. A small but significant percentage of all cancer cells are considered stem-like cells, based on their self-renewal and tumor-initiating properties. Cancer stem cells (CSCs) are resistant to standard chemotherapy and are therefore implicated in disease progression, recurrence, and adverse patient outcomes. Evidence now points to a CSC-driven hierarchal model of tumor growth, contributing to intra-tumoral heterogeneity and differential responses to chemotherapy. Hence, the need of the hour is to reproducibly identify and characterize CSC populations as well as understand the mechanisms that govern their differentiation and pro-metastatic function(s). Several studies have elucidated cellular signaling pathways important to the maintenance of cancer cell stemness; however, the study of metabolic pathways in the regulation of stemness is in its infancy. One of the few indications of CSC metabolic regulation was found in basallike breast cancer, where the transition of cells along the EMT continuum induces aerobic glycolysis and suppresses ROS production. Switching to glycolysis enhances the cells’ CSC characteristics and allows them to withstand hypoxia in the malignant environment (Dong et al., 2013). In this issue of Cell Stem Cell, Li et al. (2017) provide new insights into the interdependency of metabolism and stem cell characteristics by comparing the lipid content and composition of ALDH+ CD133+ ovarian CSCs with ALDH CD133 cancer cells. In an elegantly performed study, the authors show that ovarian CSCs and cancer cells grown as spheroids have a higher ratio of unsaturated to saturated fatty acids and that
this ratio is essential for the cells to retain stemness (Figure 1). To perform the study, Li et al. use single-cell stimulated Raman scattering (SRS) microscopy to compare the lipid content and the levels of unsaturation in the intracellular lipid pools of living single cells. By scanning single cells with a femtosecond laser and measuring Raman shifts pertaining to lipid C-H vibrations, the authors show that ovarian CSCs have significantly higher levels of lipid droplets and that these droplets contain a higher percentage of unsaturated lipids. They then go on to confirm these findings by using primary cancer cells isolated from ascites and grown as spheroids.
Given that spheroid culture increases cellular stem-like properties, their data suggest that increases in lipid unsaturation might be a general marker for CSCs in ovarian cancer. In order to determine whether lipid characteristics may be a more generalizable marker for CSC populations, it will be necessary to test multiple CSC populations from different cancers, each isolated using separate cell surface markers. However, the data presented herein strongly suggest that fatty acid desaturation is a metabolic marker of ovarian CSC populations. Fatty acid desaturases are a class of enzymes that create double bonds at
virtual energy state O
9
OH
10
saturated lipids SCD-1 O 9 10
vibrational energy state
OH
unsaturated lipids NF-κB
SCD -1 ALDH1A1
stemness
Figure 1. Higher Unsaturated Lipid Profile of Ovarian Cancer Stem Cells Attributed to Increased SCD-1 Expression Changes in lipid profile are detected using stimulated Raman scattering (SRS) microscopy. Ovarian cancer cells grown as spheroids have a higher lipid content, especially unsaturated lipids, which scatter monochromatic light differently (left panel) as depicted in the energy level diagram. The authors use this method and show that ovarian cancer stem cells (CSCs) (ALDH+/CD133+) have increased lipid droplets. Growing ovarian CSCs and cell lines as spheroids increases their unsaturated lipid content, which activates NFkB activity, thereby increasing SCD-1 and ALDH1A1 expression. These proteins subsequently increase and maintain stemness of cancer cells, thus inducing a feedforward loop where unsaturated fatty acids increase SCD-1 via NFkB, leading to enhanced unsaturated fatty acid production.
Cell Stem Cell 20, March 2, 2017 ª 2017 Elsevier Inc. 291
Cell Stem Cell
Previews specific locations in long chain fatty acids. For example palmitic acid (16:0) becomes palmitoleic acid (16:1) with a double bond at the ninth carbon position. StearoylCoA desaturase-1 (SCD-1), the most abundant desaturase, is expressed in lipogenic tissues and catalyzes the formation of double bonds at the ninth carbon atom of saturated fatty acids, leading to monounsaturated fatty acids (MUFA). Using chemical inhibitors and molecular approaches (shRNA), the authors identify SCD-1 as the enzyme responsible for the increased desaturation in stem cells. They show that blocking SCD-1 significantly reduces the stem cell population, as evidenced by inhibition of several stem cell markers, including aldehyde dehydrogenase activity, in vitro. Blocking SCD-1 in ovarian cancer spheroids before injecting them subcutaneously into mice reduces the cells’ capacity to initiate tumors and inhibits their proliferation, resulting in very small tumors. Previous reports have shown that SCD-1 is regulated by multiple oncogenic signals and that its expression correlates with cell-cycle progression and proliferation (Demoulin et al., 2004), as well as reduced apoptosis and lipid-mediated cytotoxicity (Scaglia and Igal, 2005). In addition, there is strong evidence in the literature that abnormal fatty acid composition, especially an increased MUFA to saturated fatty acid ratio, is present in several cancer types. High levels of SCD-1 and MUFA are associated with an adverse patient prognosis, especially in aggressive breast cancer (Chaje`s et al., 1999). Li et al. complements these studies by demonstrating that SCD-1 and MUFA play critical roles in the maintenance of the CSC phenotype. Moreover, the authors reinforce the view that lipids are not just a static energy depot, but rather that lipids are important modulators of the cancer phenotype. This study pro-
292 Cell Stem Cell 20, March 2, 2017
vides formative evidence that a specific lipid composition and an abundance of lipid droplets promote cancer stemness, corroborating the idea that metabolism affects every aspect of the cancer phenotype. Finally, using an unbiased approach, the authors identify several stem cellrelated pathways affected by SCD-1 inhibition. They demonstrate that NFkB signaling regulates SCD-1, lipid unsaturation, and, in consequence, stemness. It is particularly interesting that increased lipid unsaturation and active NFkB signaling upregulate ALDH1A1 mRNA levels and that NFkB inhibition abrogates this process. These findings place NFkB at the center of unsaturated lipid-mediated regulation of stemness in ovarian cancer cells. The findings also corroborate previous reports that ovarian cancer cells take up lipids from their adipocyte-rich tumor microenvironment through an FABP4dependent shuttling mechanism (Nieman et al., 2011) that is known to affect the NFkB pathway (Makowski et al., 2005). In summary, this paper is an important contribution to the CSC literature, as it demonstrates that SCD-1 is critical to maintaining the ovarian CSC phenotype by globally affecting lipogenesis. Chemotherapy is effective for non-stem cells, so SCD-1 inhibition seems to be a promising therapeutic option to specifically target stem cells and prolong patient survival. Several inhibitors of SCD-1 have been developed, mostly for diabetes treatment; however, testing has not gone beyond phase II clinical trials (Zhang et al., 2014). While the trials have not been formally published, deletion of the SCD-1 gene in a mouse model reportedly causes mechanism-based adverse effects such as narrow eye fissures, alopecia, loss of sebaceous gland function, and skin abnormalities (Miyazaki et al.,
2001), raising concerns that generalized blocking of SCD-1 may not be a clinically viable approach. However, given the convincing findings of this paper, and the urgent need for effective ovarian cancer treatments especially against stem cells, the identification of a SCD-1 inhibitor that can be clinically tolerated is clearly a worthwhile future pursuit. ACKNOWLEDGMENTS This work was supported by grants from the ovarian cancer research fund alliance (Ann and Sol Schreiber Mentored Investigator Award, awarded to A.M.) and National Cancer Institute/ National Institute of Health (R01CA169604-01A1 awarded to E.L.). We also thank Gail Isenberg (University of Chicago) for carefully editing this manuscript.
REFERENCES Chaje`s, V., Hulte´n, K., Van Kappel, A.L., Winkvist, A., Kaaks, R., Hallmans, G., Lenner, P., and Riboli, E. (1999). Int. J. Cancer 83, 585–590. Demoulin, J.B., Ericsson, J., Kallin, A., Rorsman, C., Ro¨nnstrand, L., and Heldin, C.H. (2004). J. Biol. Chem. 279, 35392–35402. Dong, C., Yuan, T., Wu, Y., Wang, Y., Fan, T.W., Miriyala, S., Lin, Y., Yao, J., Shi, J., Kang, T., et al. (2013). Cancer Cell 23, 316–331. Li, J., Condello, S., Thomes-Pepin, J., Ma, X., Xia, Y., Hurley, T.D., Matei, D., and Cheng, J.X. (2017). Cell Stem Cell 20, this issue, 303–314. Makowski, L., Brittingham, K.C., Reynolds, J.M., Suttles, J., and Hotamisligil, G.S. (2005). J. Biol. Chem. 280, 12888–12895. Miyazaki, M., Man, W.C., and Ntambi, J.M. (2001). J. Nutr. 131, 2260–2268. Nieman, K.M., Kenny, H.A., Penicka, C.V., Ladanyi, A., Buell-Gutbrod, R., Zillhardt, M.R., Romero, I.L., Carey, M.S., Mills, G.B., Hotamisligil, G.S., et al. (2011). Nat. Med. 17, 1498–1503. Scaglia, N., and Igal, R.A. (2005). J. Biol. Chem. 280, 25339–25349. Zhang, Z., Dales, N.A., and Winther, M.D. (2014). J. Med. Chem. 57, 5039–5056.