- Email: [email protected]
MUC-king with HIF May Rewire Pyrimidine Biosynthesis and Curb Gemcitabine Resistance in Pancreatic Cancer
Cancer Cell
Previews subsequent romidepsin treatment. Using enhancer cytometry from serial ATACseq measurements, the authors could deconvolve HDACi response to specific T cell subsets. Importantly, they observe a ‘‘normalizing’’ effect of HDACi on host CD4+ cells—and not on leukemic CTCL cells—that most closely correlated with the chromatin accessibility signature that differentiated normal versus CTCL T cell populations. This suggests that the clinical effects of HDACi therapy may largely be driven by immune modulation in the host T cell compartment. Further studies on larger cohorts will be required to assess the predictive or prognostic capabilities of chromatin accessibility profiling assays in CTCL, or whether specific surrogate biomarkers of HDACi response may be gleaned from these genome-wide analyses. Beyond comprehensively defining the chromatin landscapes of CTCL, the efforts of Qu et al. (2017) introduce the tantalizing prospect that personal regulome analysis may become a useful tool for the dynamic assessment of therapeutic responses in
cancer and perhaps other disease types. Many other epigenetic therapies are in use or under intense investigation for cancer treatment (targeting DNA methyltransferases and demethylases, histone methyltransferases and demethylases, bromodomains, among others); these also likely have distinct effects on chromatin accessibility signatures yet typically lack predictive biomarkers. Personal regulome profiling on broad cohorts of patients may be poised to uncover this crucial information.
Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y., and Greenleaf, W.J. (2013). Nat. Methods 10, 1213–1218.
ACKNOWLEDGMENTS
Qu, K., Zaba, L.C., Giresi, P.G., Li, R., Longmire, M., Kim, Y.H., Greenleaf, W.J., and Chang, H.Y. (2015). Cell Syst. 1, 51–61.
C.J.O. gratefully acknowledges funding support by a NCI/NIH Pathway to Independence Award K99CA190861. C.J.W. acknowledges support from NIH/NCI (1R01CA182461-02; 1R01CA184922-01, U10CA180861-01) and is a Scholar of the Leukemia and Lymphoma Society. C.J.W. is co-founder of Neon Therapeutics and a member of its scientific advisory board. REFERENCES Bolden, J.E., Peart, M.J., and Johnstone, R.W. (2006). Nat. Rev. Drug Discov. 5, 769–784.
Izban, K.F., Ergin, M., Qin, J.Z., Martinez, R.L., Pooley, R.J., Jr., Saeed, S., and Alkan, S. (2000). Hum. Pathol. 31, 1482–1490. Lee, T.I., and Young, R.A. (2013). Cell 152, 1237–1251. Lee, C.S., Ungewickell, A., Bhaduri, A., Qu, K., Webster, D.E., Armstrong, R., Weng, W.K., Aros, C.J., Mah, A., Chen, R.O., et al. (2012). Blood 120, 3288–3297. Platanias, L.C. (2005). Nat. Rev. Immunol. 5, 375–386.
Qu, K., Zaba, L.C., Satpathy, A.T., Giresi, P.G., Li, R., Jin, Y., Armstrong, R., Jin, C., Schmitt, N., Rahbar, Z.R., et al. (2017). Cancer Cell 32, this issue, 27–41. Willemze, R., Jaffe, E.S., Burg, G., Cerroni, L., Berti, E., Swerdlow, S.H., Ralfkiaer, E., Chimenti, S., Diaz-Perez, J.L., Duncan, L.M., et al. (2005). Blood 105, 3768–3785. Wolf, A.M., Wolf, D., Steurer, M., Gastl, G., Gunsilius, E., and Grubeck-Loebenstein, B. (2003). Clin. Cancer Res. 9, 606–612.
MUC-king with HIF May Rewire Pyrimidine Biosynthesis and Curb Gemcitabine Resistance in Pancreatic Cancer Chi V. Dang1,2,* 1Ludwig
Institute for Cancer Research, New York, NY 10017, USA Wistar Institute, Philadelphia, PA 19104, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ccell.2017.06.006 2The
In this issue of Cancer Cell, Singh and colleagues report a role for MUC1-induced HIF expression in rewiring ribose synthesis, which drives pyridimine production as a possible resistance mechanism to gemcitabine, adding to complexity and multiple paths to resistance. Pancreatic adenocarcinoma (PDAC) remains deadly, and its incidence is rising without effective curative treatments (Borazanci et al., 2017). Current chemotherapeutic regimens rely on gemcitabine, a cytidine analog and DNA synthesis inhibitor that is more active than 5-fluorouracil, which also interferes with DNA
synthesis (Binenbaum et al., 2015). Notwithstanding the lack of miraculous clinical responses to gemcitabine, it does provide improved survival in combination with other drugs (Binenbaum et al., 2015; Garrido-Laguna et al., 2011). However, shortly after treatment begins, gemcitabine resistance frequently emerges, blunting
therapeutic efficacy (Binenbaum et al., 2015; Rajabpour et al., 2017). In this regard, understanding resistance to gemcitabine is important to restrain this deadly disease. As such, Singh and colleagues generated gemcitabine-resistant (Gem-R) clones of the Capan and T3M4 pancreatic cancer cell lines as reagents to uncover
Cancer Cell 32, July 10, 2017 ª 2017 Elsevier Inc. 3
Cancer Cell
Previews
Figure 1. Diagram Illustrating the Transport of Glutamine, Glucose, and Gemcitabine into a PDAC Cell and the Subsequent Metabolism of These Substrates Glucose and glutamine are shown to contribute to the production of phosphoribosyl pyrophosphate (PRPP) and, via the tricarboxylic acid (TCA) cycle, aspartate that with glutamine builds on the PRPP scaffold to produce dCTP for DNA replication. Gemcitabine or 2,2-difluoro 2-deoxycytidine (dFdC) is shown metabolized to difluorodeoxycytidine triphosphate (dFCTP), which competes with dCTP, incorporates into DNA, and causes damage. HIF is depicted to stimulate the nonoxidative pentose phosphate pathway (non-ox PPP) to produce PRPP as well as stimulate selected enzymes involved in pyrimidine synthesis. Because HIF largely influences glucose metabolism, it remains open as to whether MYC, which portends poor prognosis in PDAC, could participate in resistance with its known role to directly activate many genes involved in de novo pyrimidine synthesis. Digoxin, which inhibits the expression of HIF, and leflunomide, which inhibits dihydroorotate dehydrogenase (DHODH)—an enzyme involved in de novo pyrimidine synthesis—are shown.
previously undocumented resistance mechanisms (Shukla et al., 2017). Gemcitabine (2,2-difluoro 2-deoxycytidine, dFdC) is a dideoxycytidine analog that is taken up by several cellular nucleoside transporters (Binenbaum et al., 2015). It is subsequently metabolized by deoxycytidine kinase (dCK) to difluorodeoxycytidine monophosphate (dFdCMP) and then phosphorylated to difluorodeoxycytidine triphosphate (dFdCTP), which is incorporated into DNA in competition with deoxycytidine triphosphate (dCTP) (Figure 1). That is, high levels of dCTP competitively inhibit gemcitabine incorporation and confer resistance. On the other hand, gemcitabine is inactivated by cytidine deaminase (CDA) to difluorouridine (dFdU) that is degraded and excreted. As such, decreased dCK, which is required for activation of gemcitabine, or increased CDA, which degrades gemcitabine, have been implicated as gemcitabine-resistance mechanisms (Binenbaum et al., 2015). All of these cell-intrinsic biochemical mechanisms converge on diminished 4 Cancer Cell 32, July 10, 2017
gemcitabine levels or increased endogenous dCTP levels (Binenbaum et al., 2015). To determine mechanisms of gemcitabine resistance, Singh and colleagues exposed Capan and T3M4 cell lines to 6 months of gemcitabine to generate resistant (Gem-R) clones (Shukla et al., 2017). Through steady-state metabolomics, they found differences between the parental wild-type (WT) cell line and Gem-R cells construed to suggest that the resistant cells had accentuated the non-oxidative branch of the pentose phosphate pathway activity and increased pyrimidine biosynthesis (Figure 1). These changes were hypothesized to confer resistance via increased dCTP production, which was partially corroborated by the ability of exogenous deoxycytidine to confer gemcitabine resistance. Based on these observations, the authors measured expression of genes involved in pyrimidine biosynthesis and found that many were upregulated in the Gem-R cells. The authors further gleaned from studying genes involved in pyrimidine
and purine synthesis and their correlation with gemcitabine sensitivity among 17 pancreatic cancer cell lines in vitro, leading to identification that PDAC patients with increased expression of transketolase (TKT), which is involved in the pentose phosphate pathway, and CTP synthase (CTPS) had decreased progression free survival. While these correlations support the overarching hypothesis, it is not surprising that upregulation of nucleotide synthesis genes that is sine qua non with cell proliferation would be a poor prognostic indicator, not necessarily linked to gemcitabine resistance. Hence, additional studies focused on an unbiased assessment of the entire transcriptome (through RNA-seq) could provide valuable information regarding potential pleiotropic adaptive mechanisms of the Gem-R cells as compared with WT parental cells. This information then could be used to mine the available genomic data from The Cancer Genome Atlas (TCGA) or International Cancer Genome Consortium in an unbiased manner (Bailey et al., 2016).
Cancer Cell
Previews The authors hypothesized that the hypoxia inducible factors (HIFs) could play a role in the resistance phenotype, particularly since PDAC tends to be hypoxic (Shukla et al., 2017). They provide evidence that the Gem-R cells had baseline elevated HIF-1a expression that depended on MUC1 elevated expression under normoxic conditions (Figure 1). It is unclear, however, how relevant this axis is in vivo because PDAC tends to be quite hypoxic (Borazanci et al., 2017), which would increase HIF-1a levels independent of MUC1. Specifically, the authors provide evidence of the in situ co-localization of the hypoxic marker EF5 staining with expression of carbonic anhydrase IX (CAIX), a target of HIF, and TKT and CTPS in orthotopically implanted Capan-1 cell line. These observations pose a question as to whether in vivo hypoxia would be sufficient to confer resistance to gemcitabine through induction of HIF as proposed by the authors. Notwithstanding the unresolved role of in situ hypoxia (Figure 1) versus MUC1 in the expression of HIF-1 in vivo, the authors documented that siRNA-mediated knockdown of HIF-1a diminished the resistance phenotype and rendered Gem-R cells more sensitive to gemcitabine-mediated cell death (Shukla et al., 2017). It is surmised that HIF mediates resistance through induction of phosphoribosyl pyrophosphate (PRPP) and nucleotide synthesis. Amino acid uptake is also integral for nucleotide synthesis. Specifically, de novo pyrimidine synthesis depends on PRPP, upon which carbon dioxide, aspartate, and glutamine contribute to the pyrimidine ring synthesis (Figure 1). HIF activates largely glucose and not glutamine metabolism. By contrast, MYC could stimulate glutamine metabolism and global transcription of genes involved in pyrimidine biosynthesis, which is further increased through activation by mTOR (Ben-Sahra et al., 2013; Liu et al., 2008; Mannava et al., 2008) (Figure 1). Further,
MYC expression has been documented in genome-wide studies to be associated with poor prognosis in PDAC (Wirth et al., 2016). Thus, while Singh and colleagues demonstrate that HIF may be necessary for the resistance phenotype, additional studies should address whether HIF, via overexpression of a constitutively stabilized HIF-1a mutant, is sufficient for resistance or whether activation of additional metabolic mediators is required to confer gemcitabine resistance. Based on the observation that pyrimidine biosynthesis and HIF appear to confer resistance to gemcitabine, the authors used leflunomide, an immunosuppressant and inhibitor of dihydroorotate dehydrogenase (DHODH, which is essential for de novo pyrimidine synthesis) or digoxin to decrease HIF levels and found that both could trigger gemcitabine toxicity to Gem-R cells in vitro and slowed orthotopic xenograft growth in vivo (Figure 1). It would be interesting to determine whether leflunomide or digoxin would be synergistic with other therapeutic agents independent of the hypothesized mechanism of neutralizing the Gem-R phenotype. This is particularly important since inhibition of pyrimidine synthesis by leflunomide or HIF by digoxin would disable DNA synthesis and adaptation to hypoxia, presumably rendering pancreatic cancer cells sensitive to any therapeutic stress and, hence, death. In addition to cell-autonomous mechanisms of gemcitabine resistance, the PDAC tumor microenvironment also appears to contribute to resistance. In this regard, one study revealed that Gem-R tumors have an enrichment of stromal gene expression among a panel of 23 PDAC patient-derived xenografts (PDXs), indicating the possible role of the stroma in addition to cell-autonomous mechanisms of gemcitabine resistance (Garrido-Laguna et al., 2011). Future studies of a panel of PDAC PDXs
or genetically engineered models on PDAC in response to gemcitabine and digoxin or leflunomide would be highly instructive. Overall, while there are still open questions regarding the model proposed by the authors as discussed, this paper nicely contributes to our understanding of the complexity of the biology of pancreatic cancer and opens new avenues for investigation. ACKNOWLEDGMENTS Our original research is supported by the National Cancer Institute grants CA051497 and CA057341 and Stand Up To Cancer grant SU2C-AACR- 395 DT0509. REFERENCES Bailey, P., Chang, D.K., Nones, K., Johns, A.L., Patch, A.M., Gingras, M.C., Miller, D.K., Christ, A.N., Bruxner, T.J., Quinn, M.C., et al.; Australian Pancreatic Cancer Genome Initiative (2016). Nature 531, 47–52. Ben-Sahra, I., Howell, J.J., Asara, J.M., and Manning, B.D. (2013). Science 339, 1323–1328. Binenbaum, Y., Na’ara, S., and Gil, Z. (2015). Drug Resist. Updat. 23, 55–68. Borazanci, E., Dang, C.V., Robey, R.W., Bates, S.E., Chabot, J.A., and Von Hoff, D.D. (2017). Clin. Cancer Res. 23, 1629–1637. Garrido-Laguna, I., Uson, M., Rajeshkumar, N.V., Tan, A.C., de Oliveira, E., Karikari, C., Villaroel, M.C., Salomon, A., Taylor, G., Sharma, R., et al. (2011). Clin. Cancer Res. 17, 5793–5800. Liu, Y.C., Li, F., Handler, J., Huang, C.R., Xiang, Y., Neretti, N., Sedivy, J.M., Zeller, K.I., and Dang, C.V. (2008). PLoS One 3, e2722. Mannava, S., Grachtchouk, V., Wheeler, L.J., Im, M., Zhuang, D., Slavina, E.G., Mathews, C.K., Shewach, D.S., and Nikiforov, M.A. (2008). Cell Cycle 7, 2392–2400. Rajabpour, A., Rajaei, F., and Teimoori-Toolabi, L. (2017). Pancreatology 17, 310–320. Shukla, S.K., Purohit, V., Mehla, K., Gunda, V., Chaika, N., Vernucci, E., King, R.J., Abrego, J., Goode, G.D., Dasgupta, A., et al. (2017). Cancer Cell 32, this issue, 71–87. €mer, O.H., and Wirth, M., Mahboobi, S., Kra Schneider, G. (2016). Mol. Cancer Ther. 15, 1792–1798.
Cancer Cell 32, July 10, 2017 5
Previews subsequent romidepsin treatment. Using enhancer cytometry from serial ATACseq measurements, the authors could deconvolve HDACi response to specific T cell subsets. Importantly, they observe a ‘‘normalizing’’ effect of HDACi on host CD4+ cells—and not on leukemic CTCL cells—that most closely correlated with the chromatin accessibility signature that differentiated normal versus CTCL T cell populations. This suggests that the clinical effects of HDACi therapy may largely be driven by immune modulation in the host T cell compartment. Further studies on larger cohorts will be required to assess the predictive or prognostic capabilities of chromatin accessibility profiling assays in CTCL, or whether specific surrogate biomarkers of HDACi response may be gleaned from these genome-wide analyses. Beyond comprehensively defining the chromatin landscapes of CTCL, the efforts of Qu et al. (2017) introduce the tantalizing prospect that personal regulome analysis may become a useful tool for the dynamic assessment of therapeutic responses in
cancer and perhaps other disease types. Many other epigenetic therapies are in use or under intense investigation for cancer treatment (targeting DNA methyltransferases and demethylases, histone methyltransferases and demethylases, bromodomains, among others); these also likely have distinct effects on chromatin accessibility signatures yet typically lack predictive biomarkers. Personal regulome profiling on broad cohorts of patients may be poised to uncover this crucial information.
Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y., and Greenleaf, W.J. (2013). Nat. Methods 10, 1213–1218.
ACKNOWLEDGMENTS
Qu, K., Zaba, L.C., Giresi, P.G., Li, R., Longmire, M., Kim, Y.H., Greenleaf, W.J., and Chang, H.Y. (2015). Cell Syst. 1, 51–61.
C.J.O. gratefully acknowledges funding support by a NCI/NIH Pathway to Independence Award K99CA190861. C.J.W. acknowledges support from NIH/NCI (1R01CA182461-02; 1R01CA184922-01, U10CA180861-01) and is a Scholar of the Leukemia and Lymphoma Society. C.J.W. is co-founder of Neon Therapeutics and a member of its scientific advisory board. REFERENCES Bolden, J.E., Peart, M.J., and Johnstone, R.W. (2006). Nat. Rev. Drug Discov. 5, 769–784.
Izban, K.F., Ergin, M., Qin, J.Z., Martinez, R.L., Pooley, R.J., Jr., Saeed, S., and Alkan, S. (2000). Hum. Pathol. 31, 1482–1490. Lee, T.I., and Young, R.A. (2013). Cell 152, 1237–1251. Lee, C.S., Ungewickell, A., Bhaduri, A., Qu, K., Webster, D.E., Armstrong, R., Weng, W.K., Aros, C.J., Mah, A., Chen, R.O., et al. (2012). Blood 120, 3288–3297. Platanias, L.C. (2005). Nat. Rev. Immunol. 5, 375–386.
Qu, K., Zaba, L.C., Satpathy, A.T., Giresi, P.G., Li, R., Jin, Y., Armstrong, R., Jin, C., Schmitt, N., Rahbar, Z.R., et al. (2017). Cancer Cell 32, this issue, 27–41. Willemze, R., Jaffe, E.S., Burg, G., Cerroni, L., Berti, E., Swerdlow, S.H., Ralfkiaer, E., Chimenti, S., Diaz-Perez, J.L., Duncan, L.M., et al. (2005). Blood 105, 3768–3785. Wolf, A.M., Wolf, D., Steurer, M., Gastl, G., Gunsilius, E., and Grubeck-Loebenstein, B. (2003). Clin. Cancer Res. 9, 606–612.
MUC-king with HIF May Rewire Pyrimidine Biosynthesis and Curb Gemcitabine Resistance in Pancreatic Cancer Chi V. Dang1,2,* 1Ludwig
Institute for Cancer Research, New York, NY 10017, USA Wistar Institute, Philadelphia, PA 19104, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ccell.2017.06.006 2The
In this issue of Cancer Cell, Singh and colleagues report a role for MUC1-induced HIF expression in rewiring ribose synthesis, which drives pyridimine production as a possible resistance mechanism to gemcitabine, adding to complexity and multiple paths to resistance. Pancreatic adenocarcinoma (PDAC) remains deadly, and its incidence is rising without effective curative treatments (Borazanci et al., 2017). Current chemotherapeutic regimens rely on gemcitabine, a cytidine analog and DNA synthesis inhibitor that is more active than 5-fluorouracil, which also interferes with DNA
synthesis (Binenbaum et al., 2015). Notwithstanding the lack of miraculous clinical responses to gemcitabine, it does provide improved survival in combination with other drugs (Binenbaum et al., 2015; Garrido-Laguna et al., 2011). However, shortly after treatment begins, gemcitabine resistance frequently emerges, blunting
therapeutic efficacy (Binenbaum et al., 2015; Rajabpour et al., 2017). In this regard, understanding resistance to gemcitabine is important to restrain this deadly disease. As such, Singh and colleagues generated gemcitabine-resistant (Gem-R) clones of the Capan and T3M4 pancreatic cancer cell lines as reagents to uncover
Cancer Cell 32, July 10, 2017 ª 2017 Elsevier Inc. 3
Cancer Cell
Previews
Figure 1. Diagram Illustrating the Transport of Glutamine, Glucose, and Gemcitabine into a PDAC Cell and the Subsequent Metabolism of These Substrates Glucose and glutamine are shown to contribute to the production of phosphoribosyl pyrophosphate (PRPP) and, via the tricarboxylic acid (TCA) cycle, aspartate that with glutamine builds on the PRPP scaffold to produce dCTP for DNA replication. Gemcitabine or 2,2-difluoro 2-deoxycytidine (dFdC) is shown metabolized to difluorodeoxycytidine triphosphate (dFCTP), which competes with dCTP, incorporates into DNA, and causes damage. HIF is depicted to stimulate the nonoxidative pentose phosphate pathway (non-ox PPP) to produce PRPP as well as stimulate selected enzymes involved in pyrimidine synthesis. Because HIF largely influences glucose metabolism, it remains open as to whether MYC, which portends poor prognosis in PDAC, could participate in resistance with its known role to directly activate many genes involved in de novo pyrimidine synthesis. Digoxin, which inhibits the expression of HIF, and leflunomide, which inhibits dihydroorotate dehydrogenase (DHODH)—an enzyme involved in de novo pyrimidine synthesis—are shown.
previously undocumented resistance mechanisms (Shukla et al., 2017). Gemcitabine (2,2-difluoro 2-deoxycytidine, dFdC) is a dideoxycytidine analog that is taken up by several cellular nucleoside transporters (Binenbaum et al., 2015). It is subsequently metabolized by deoxycytidine kinase (dCK) to difluorodeoxycytidine monophosphate (dFdCMP) and then phosphorylated to difluorodeoxycytidine triphosphate (dFdCTP), which is incorporated into DNA in competition with deoxycytidine triphosphate (dCTP) (Figure 1). That is, high levels of dCTP competitively inhibit gemcitabine incorporation and confer resistance. On the other hand, gemcitabine is inactivated by cytidine deaminase (CDA) to difluorouridine (dFdU) that is degraded and excreted. As such, decreased dCK, which is required for activation of gemcitabine, or increased CDA, which degrades gemcitabine, have been implicated as gemcitabine-resistance mechanisms (Binenbaum et al., 2015). All of these cell-intrinsic biochemical mechanisms converge on diminished 4 Cancer Cell 32, July 10, 2017
gemcitabine levels or increased endogenous dCTP levels (Binenbaum et al., 2015). To determine mechanisms of gemcitabine resistance, Singh and colleagues exposed Capan and T3M4 cell lines to 6 months of gemcitabine to generate resistant (Gem-R) clones (Shukla et al., 2017). Through steady-state metabolomics, they found differences between the parental wild-type (WT) cell line and Gem-R cells construed to suggest that the resistant cells had accentuated the non-oxidative branch of the pentose phosphate pathway activity and increased pyrimidine biosynthesis (Figure 1). These changes were hypothesized to confer resistance via increased dCTP production, which was partially corroborated by the ability of exogenous deoxycytidine to confer gemcitabine resistance. Based on these observations, the authors measured expression of genes involved in pyrimidine biosynthesis and found that many were upregulated in the Gem-R cells. The authors further gleaned from studying genes involved in pyrimidine
and purine synthesis and their correlation with gemcitabine sensitivity among 17 pancreatic cancer cell lines in vitro, leading to identification that PDAC patients with increased expression of transketolase (TKT), which is involved in the pentose phosphate pathway, and CTP synthase (CTPS) had decreased progression free survival. While these correlations support the overarching hypothesis, it is not surprising that upregulation of nucleotide synthesis genes that is sine qua non with cell proliferation would be a poor prognostic indicator, not necessarily linked to gemcitabine resistance. Hence, additional studies focused on an unbiased assessment of the entire transcriptome (through RNA-seq) could provide valuable information regarding potential pleiotropic adaptive mechanisms of the Gem-R cells as compared with WT parental cells. This information then could be used to mine the available genomic data from The Cancer Genome Atlas (TCGA) or International Cancer Genome Consortium in an unbiased manner (Bailey et al., 2016).
Cancer Cell
Previews The authors hypothesized that the hypoxia inducible factors (HIFs) could play a role in the resistance phenotype, particularly since PDAC tends to be hypoxic (Shukla et al., 2017). They provide evidence that the Gem-R cells had baseline elevated HIF-1a expression that depended on MUC1 elevated expression under normoxic conditions (Figure 1). It is unclear, however, how relevant this axis is in vivo because PDAC tends to be quite hypoxic (Borazanci et al., 2017), which would increase HIF-1a levels independent of MUC1. Specifically, the authors provide evidence of the in situ co-localization of the hypoxic marker EF5 staining with expression of carbonic anhydrase IX (CAIX), a target of HIF, and TKT and CTPS in orthotopically implanted Capan-1 cell line. These observations pose a question as to whether in vivo hypoxia would be sufficient to confer resistance to gemcitabine through induction of HIF as proposed by the authors. Notwithstanding the unresolved role of in situ hypoxia (Figure 1) versus MUC1 in the expression of HIF-1 in vivo, the authors documented that siRNA-mediated knockdown of HIF-1a diminished the resistance phenotype and rendered Gem-R cells more sensitive to gemcitabine-mediated cell death (Shukla et al., 2017). It is surmised that HIF mediates resistance through induction of phosphoribosyl pyrophosphate (PRPP) and nucleotide synthesis. Amino acid uptake is also integral for nucleotide synthesis. Specifically, de novo pyrimidine synthesis depends on PRPP, upon which carbon dioxide, aspartate, and glutamine contribute to the pyrimidine ring synthesis (Figure 1). HIF activates largely glucose and not glutamine metabolism. By contrast, MYC could stimulate glutamine metabolism and global transcription of genes involved in pyrimidine biosynthesis, which is further increased through activation by mTOR (Ben-Sahra et al., 2013; Liu et al., 2008; Mannava et al., 2008) (Figure 1). Further,
MYC expression has been documented in genome-wide studies to be associated with poor prognosis in PDAC (Wirth et al., 2016). Thus, while Singh and colleagues demonstrate that HIF may be necessary for the resistance phenotype, additional studies should address whether HIF, via overexpression of a constitutively stabilized HIF-1a mutant, is sufficient for resistance or whether activation of additional metabolic mediators is required to confer gemcitabine resistance. Based on the observation that pyrimidine biosynthesis and HIF appear to confer resistance to gemcitabine, the authors used leflunomide, an immunosuppressant and inhibitor of dihydroorotate dehydrogenase (DHODH, which is essential for de novo pyrimidine synthesis) or digoxin to decrease HIF levels and found that both could trigger gemcitabine toxicity to Gem-R cells in vitro and slowed orthotopic xenograft growth in vivo (Figure 1). It would be interesting to determine whether leflunomide or digoxin would be synergistic with other therapeutic agents independent of the hypothesized mechanism of neutralizing the Gem-R phenotype. This is particularly important since inhibition of pyrimidine synthesis by leflunomide or HIF by digoxin would disable DNA synthesis and adaptation to hypoxia, presumably rendering pancreatic cancer cells sensitive to any therapeutic stress and, hence, death. In addition to cell-autonomous mechanisms of gemcitabine resistance, the PDAC tumor microenvironment also appears to contribute to resistance. In this regard, one study revealed that Gem-R tumors have an enrichment of stromal gene expression among a panel of 23 PDAC patient-derived xenografts (PDXs), indicating the possible role of the stroma in addition to cell-autonomous mechanisms of gemcitabine resistance (Garrido-Laguna et al., 2011). Future studies of a panel of PDAC PDXs
or genetically engineered models on PDAC in response to gemcitabine and digoxin or leflunomide would be highly instructive. Overall, while there are still open questions regarding the model proposed by the authors as discussed, this paper nicely contributes to our understanding of the complexity of the biology of pancreatic cancer and opens new avenues for investigation. ACKNOWLEDGMENTS Our original research is supported by the National Cancer Institute grants CA051497 and CA057341 and Stand Up To Cancer grant SU2C-AACR- 395 DT0509. REFERENCES Bailey, P., Chang, D.K., Nones, K., Johns, A.L., Patch, A.M., Gingras, M.C., Miller, D.K., Christ, A.N., Bruxner, T.J., Quinn, M.C., et al.; Australian Pancreatic Cancer Genome Initiative (2016). Nature 531, 47–52. Ben-Sahra, I., Howell, J.J., Asara, J.M., and Manning, B.D. (2013). Science 339, 1323–1328. Binenbaum, Y., Na’ara, S., and Gil, Z. (2015). Drug Resist. Updat. 23, 55–68. Borazanci, E., Dang, C.V., Robey, R.W., Bates, S.E., Chabot, J.A., and Von Hoff, D.D. (2017). Clin. Cancer Res. 23, 1629–1637. Garrido-Laguna, I., Uson, M., Rajeshkumar, N.V., Tan, A.C., de Oliveira, E., Karikari, C., Villaroel, M.C., Salomon, A., Taylor, G., Sharma, R., et al. (2011). Clin. Cancer Res. 17, 5793–5800. Liu, Y.C., Li, F., Handler, J., Huang, C.R., Xiang, Y., Neretti, N., Sedivy, J.M., Zeller, K.I., and Dang, C.V. (2008). PLoS One 3, e2722. Mannava, S., Grachtchouk, V., Wheeler, L.J., Im, M., Zhuang, D., Slavina, E.G., Mathews, C.K., Shewach, D.S., and Nikiforov, M.A. (2008). Cell Cycle 7, 2392–2400. Rajabpour, A., Rajaei, F., and Teimoori-Toolabi, L. (2017). Pancreatology 17, 310–320. Shukla, S.K., Purohit, V., Mehla, K., Gunda, V., Chaika, N., Vernucci, E., King, R.J., Abrego, J., Goode, G.D., Dasgupta, A., et al. (2017). Cancer Cell 32, this issue, 71–87. €mer, O.H., and Wirth, M., Mahboobi, S., Kra Schneider, G. (2016). Mol. Cancer Ther. 15, 1792–1798.
Cancer Cell 32, July 10, 2017 5