Diablo: A Double-Edged Sword in Cancer?
Authors: Jack L. Arbiser
Journal: Molecular Therapy (2018)

SMAC/Diablo is known to be a pro-apoptotic protein and its overexpression has been observed to oppose progression of a variety of tumor types. However, in this issue of Molecular Therapy, Paul et al.1Paul A. Krelin Y. Arif T. Jeger R. Shoshan-Barmatz V. A new role for the mitochondrial pro-apoptotic protein SMAC/Diablo in phospholipid synthesis associated with tumorigenesis.Mol Ther. 2018; 26 (this issue): 680-694Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar reveal a new function for the protein, suggesting that the role of SMAC/Diablo in cancer can be a double-edged sword. The authors were puzzled by the increased expression of SMAC/Diablo in multiple types of cancer, including solid tumors such as lung, breast, gastric chronic lymphocytic leukemia, and B cell lymphoma. In addition, nuclear expression of this normally mitochondrial protein was observed in some malignancies. Therefore, they reasoned that there must be additional functions of SMAC/Diablo, and that the current dogma of it being purely anti-apoptotic is perhaps too simplistic. Their findings show that SMAC/Diablo possesses a new lipid synthesis-related function essential for cancer growth, rendering SMAC/Diablo a promising therapeutic target in cancer. SMAC/DIABLO is a mitochondrial protein that can enhance apoptosis, possibly by neutralizing the caspase-inhibitory properties of members of the IAP family of proteins. It is encoded by a nuclear gene and is subsequently imported into mitochondria, at which time its N terminus is removed by proteolysis to generate the mature protein. SMAC/DIABLO has been implicated in tumor development owing to its proapoptotic effects, and small molecule effectors have been developed as a cancer treatment.2Petersen S.L. Wang L. Yalcin-Chin A. Li L. Peyton M. Minna J. Harran P. Wang X. Autocrine TNFalpha signaling renders human cancer cells susceptible to Smac-mimetic-induced apoptosis.Cancer Cell. 2007; 12: 445-456Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar, 3Cuvillier O. Levade T. Sphingosine 1-phosphate antagonizes apoptosis of human leukemia cells by inhibiting release of cytochrome c and Smac/DIABLO from mitochondria.Blood. 2001; 98: 2828-2836Crossref PubMed Scopus (117) Google Scholar, 4Petersen S.L. Peyton M. Minna J.D. Wang X. Overcoming cancer cell resistance to Smac mimetic induced apoptosis by modulating cIAP-2 expression.Proc. Natl. Acad. Sci. USA. 2010; 107: 11936-11941Crossref PubMed Scopus (94) Google Scholar However, in a recent study of non-small cell lung carcinoma, the Shoshan-Bartmatz group5Shoshan-Barmatz V. Bishitz Y. Paul A. Krelin Y. Nakdimon I. Peled N. Lavon A. Rudoy-Zilberman E. Refaely Y. A molecular signature of lung cancer: potential biomarkers for adenocarcinoma and squamous cell carcinoma.Oncotarget. 2017; 8: 105492-105509Crossref PubMed Scopus (20) Google Scholar demonstrated high expression of SMAC/DIABLO in squamous cell carcinoma (SCC) of the lung compared to adenocarcinoma. Of interest, SCC is thought to derive from carcinogen-induced metaplasia. Similarly, Barrett’s esophagus, a metaplastic change of esophageal epithelium and precursor of esophageal carcinoma, also demonstrated elevated levels of SMAC/DIABLO.6O’Farrell N.J. Feighery R. Picardo S.L. Lynam-Lennon N. Biniecka M. McGarrigle S.A. Phelan J.J. MacCarthy F. O’Toole D. Fox E.J. et al.Changes in mitochondrial stability during the progression of the Barrett’s esophagus disease sequence.BMC Cancer. 2016; 16: 497Crossref PubMed Scopus (11) Google Scholar These findings suggest that SMAC/DIABLO overexpression may be necessary to promote neoplastic metaplasia. Examination of a series of human cell lines revealed a general upregulation of SMAC/Diablo in all malignant cell lines, despite differences in p53, p16, and Kras status. To explore further the role of SMAC/DIABLO in tumor cells, the authors introduced short inhibitory RNA (siRNA) specific to the human gene (si-hSMAC-A) into a series of cell lines and showed that they could successfully knock down protein levels. Interestingly, all of the cell lines in which SMAC/Diablo was knocked down demonstrated reduced rates of proliferation, but not increased levels of apoptosis. It may be that cancer cells have to maintain SMAC/DIABLO expression in an optimal range, with increased expression with respect to this optimum leading to apoptosis/ferroptosis, and reduced expression inducing differentiation. Stable siRNA knockdown of SMAC/Diablo also led to decreased tumor growth in vivo, again primarily due to a proliferative defect rather than increased apoptosis, consistent with the in vitro data. Gene array analysis of SMAC/Diablo knockdown cells revealed profound changes of endoplasmic reticulum and Golgi related genes, metabolism, and differentiation associated genes. Of note, alveolar differentiation to A1 cells was noted in A549 lung cancer cells in which SMAC/DIABLO was downregulated, indicating that SMAC/DIABLO may act as an inhibitor of differentiation. Several questions arise: first, do increased levels of SMAC/DIABLO repress the expression of differentiation associated transcription factors, or merely downregulate some of their targets? For instance, there are several transcription factors that are associated with differentiation, such as microphthalmia (MITF) and PPAR gamma (PPARg), which are required to the differentiation of melanocytes and adipocytes, respectively, but can also act as lineage specific oncogenes.7Vazquez F. Lim J.H. Chim H. Bhalla K. Girnun G. Pierce K. Clish C.B. Granter S.R. Widlund H.R. Spiegelman B.M. Puigserver P. PGC1α expression defines a subset of human melanoma tumors with increased mitochondrial capacity and resistance to oxidative stress.Cancer Cell. 2013; 23: 287-301Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 8Pleniceanu O. Shukrun R. Omer D. Vax E. Kanter I. Dziedzic K. Pode-Shakked N. Mark-Daniel M. Pri-Chen S. Gnatek Y. et al.PPARG is central to the initiation and propagation of human angiomyolipoma, suggesting its potential as a therapeutic target.EMBO Mol. Med. 2017; 9: 508-530Crossref PubMed Scopus (10) Google Scholar Second, what is the metabolic phenotype in terms of respiration or glycolysis? Tumor cells typically switch from cellular respiration to the very inefficient glycolysis for their ATP needs. The authors observed that downregulation of SMAC/DIABLO led to a reduction of ATP by 25%–30%, but this does not mean that the cells were necessarily glycolytic, as more differentiated cells may have lower ATP needs. Indeed, the proliferative defect in knocked down cells was far greater than the loss of ATP, and there was minimal cell death. This disparity suggests induction of differentiation rather than a simple cell cycle block or induction of apoptosis. Interestingly, the gene most affected by knockdown of SMAC/DIABLO is Elovl4. Elovl4 encodes an enzyme catalyzing elongation of fatty acids and is mutated in the human disease Stargardts macular dystrophy.9Zhang K. Kniazeva M. Han M. Li W. Yu Z. Yang Z. Li Y. Metzker M.L. Allikmets R. Zack D.J. et al.A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.Nat. Genet. 2001; 27: 89-93Crossref PubMed Scopus (376) Google Scholar Loss of Elovl4 results in loss of very long chain polyunsaturated fatty acid (VLC-PUFA), which is required for optimal retinal and skin function.10Westerberg R. Månsson J.E. Golozoubova V. Shabalina I.G. Backlund E.C. Tvrdik P. Retterstøl K. Capecchi M.R. Jacobsson A. ELOVL3 is an important component for early onset of lipid recruitment in brown adipose tissue.J. Biol. Chem. 2006; 281: 4958-4968Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 11Li W. Sandhoff R. Kono M. Zerfas P. Hoffmann V. Ding B.C. Proia R.L. Deng C.X. Depletion of ceramides with very long chain fatty acids causes defective skin permeability barrier function, and neonatal lethality in ELOVL4 deficient mice.Int. J. Biol. Sci. 2007; 3: 120-128Crossref PubMed Scopus (128) Google Scholar Knockout of Elovl4 results in lethality due to a skin phenotype.11Li W. Sandhoff R. Kono M. Zerfas P. Hoffmann V. Ding B.C. Proia R.L. Deng C.X. Depletion of ceramides with very long chain fatty acids causes defective skin permeability barrier function, and neonatal lethality in ELOVL4 deficient mice.Int. J. Biol. Sci. 2007; 3: 120-128Crossref PubMed Scopus (128) Google Scholar Interestingly, Elovl4 is hypermethylated in cancer, and thus the products of Elovl4 may be tumor suppressive.12Revill K. Wang T. Lachenmayer A. Kojima K. Harrington A. Li J. Hoshida Y. Llovet J.M. Powers S. Genome-wide methylation analysis and epigenetic unmasking identify tumor suppressor genes in hepatocellular carcinoma.Gastroenterology. 2013; 145: 1424-1435Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 13Moon J.W. Lee S.K. Lee J.O. Kim N. Lee Y.W. Kim S.J. Kang H.J. Kim J. Kim H.S. Park S.H. Identification of novel hypermethylated genes and demethylating effect of vincristine in colorectal cancer.J. Exp. Clin. Cancer Res. 2014; 33: 4Crossref PubMed Scopus (34) Google Scholar The authors showed that phospholipid synthesis was globally inhibited by SMAC/DIABLO depletion, accompanied by changes in the extracellular matrix and a reduction in the number of infiltrating myofibroblasts in the knockdown tumors. Novel small molecules termed elovanoids, which are very long fatty acid derivatives,14Jun B. Mukherjee P.K. Asatryan A. Kautzmann M.A. Heap J. Gordon W.C. Bhattacharjee S. Yang R. Petasis N.A. Bazan N.G. Elovanoids are novel cell-specific lipid mediators necessary for neuroprotective signaling for photoreceptor cell integrity.Sci. Rep. 2017; 7: 5279Crossref PubMed Scopus (41) Google Scholar have been synthesized and can be used to determine whether supplementation causes the same effect as SMAC/DIABLO knockout or is necessary for preserving the phenotype of the more differentiated tumor cell after SMAC/DIABLO knockout. This could indicate a decrease in TGF beta signaling, and could be of benefit in tumors that are desmoplastic, such as pancreatic carcinoma, which is notorious for its desmoplasia.15Lee J.J. Perera R.M. Wang H. Wu D.C. Liu X.S. Han S. Fitamant J. Jones P.D. Ghanta K.S. Kawano S. et al.Stromal response to Hedgehog signaling restrains pancreatic cancer progression.Proc. Natl. Acad. Sci. USA. 2014; 111: E3091-E3100Crossref PubMed Scopus (360) Google Scholar, 16Gore J. Korc M. Pancreatic cancer stroma: friend or foe?.Cancer Cell. 2014; 25: 711-712Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar Desmoplasia is the growth of fibrous or connective tissue that may occur around a neoplasm, causing dense fibrosis around the tumor. The decreased phospholipid synthesis in the SMAC/DIABLO knockout may translate into decreased concentrations of sphingosine-1 phosphate, which is essential for TGF beta-induced desmoplasia.17Gendron D.R. Lemay A.M. Lecours P.B. Perreault-Vallières V. Huppé C.A. Bossé Y. Blanchet M.R. Dion G. Marsolais D. FTY720 promotes pulmonary fibrosis when administered during the remodelling phase following a bleomycin-induced lung injury.Pulm. Pharmacol. Ther. 2017; 44: 50-56Crossref PubMed Scopus (11) Google Scholar, 18Yamaguchi H. Kitayama J. Takuwa N. Arikawa K. Inoki I. Takehara K. Nagawa H. Takuwa Y. Sphingosine-1-phosphate receptor subtype-specific positive and negative regulation of Rac and haematogenous metastasis of melanoma cells.Biochem. J. 2003; 374: 715-722Crossref PubMed Scopus (89) Google Scholar Finally, ceramide analogs that cannot be metabolized to sphingosine-1 phosphate are available to help dissect the balance of ceramide and the ceramide metabolite sphingosine-1 phosphate.19Arbiser J.L. Nowak R. Michaels K. Skabytska Y. Biedermann T. Lewis M.J. Bonner M.Y. Rao S. Gilbert L.C. Yusuf N. et al.Evidence for biochemical barrier restoration: Topical solenopsin analogs improve inflammation and acanthosis in the KC-Tie2 mouse model of psoriasis.Sci. Rep. 2017; 7: 11198Crossref PubMed Scopus (10) Google Scholar Drugs that downregulate SMAC/DIABLO might modify the tumor microenvironment to decrease desmoplasia so as to potentiate chemotherapy and radiation. Induction of SMAC/DIABLO has been seen as a potential target for inducing tumor apoptosis. The studies of Paul et al.1Paul A. Krelin Y. Arif T. Jeger R. Shoshan-Barmatz V. A new role for the mitochondrial pro-apoptotic protein SMAC/Diablo in phospholipid synthesis associated with tumorigenesis.Mol Ther. 2018; 26 (this issue): 680-694Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar suggest the opposite: blockade of SMAC/DIABLO might induce a proliferation block through induction of differentiation. Therefore, the blockade of SMAC/DIABLO with a small molecule might represent a novel therapeutic modality through a coordinate regulation of phospholipid synthesis and decreased tumor desmoplasia. Successful treatment of cancers may involve sequential use of Smac mimetics to kill tumors through apoptosis/ferroptosis, alternating with Smac inhibitors that can promote differentiation and exit from cell cycle. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Dr. Arbiser is supported by NIH R01 AR47901. A New Role for the Mitochondrial Pro-apoptotic Protein SMAC/Diablo in Phospholipid Synthesis Associated with TumorigenesisPaul et al.Molecular TherapyDecember 22, 2017In BriefParadoxically, the mitochondrial pro-apoptotic protein SMAC/Diablo is overexpressed in several cancers. Silencing SMAC/Diablo expression in cancer cells and in mice lung cancer xenografts reduced cell and tumor growth and phospholipids levels. We demonstrate that SMAC/Diablo possesses a new lipid synthesis-related function essential for cancer growth, making SMAC/Diablo a promising therapeutic target. Full-Text PDF Open Archive