Cancer cells exhibit an aberrant metabolism that facilitates more efficient production of biomass and hence tumor growth and progression. However, the genetic cues modulating this metabolic switch remain largely undetermined. We identified a metabolic function for the promyelocytic leukemia (PML) gene, uncovering an unexpected role for this bona fide tumor suppressor in breast cancer cell survival. We found that PML acted as both a negative regulator of PPARγ coactivator 1A (PGC1A) acetylation and a potent activator of PPAR signaling and fatty acid oxidation. We further showed that PML promoted ATP production and inhibited anoikis. Importantly, PML expression allowed luminal filling in 3D basement membrane breast culture models, an effect that was reverted by the pharmacological inhibition of fatty acid oxidation. Additionally, immunohistochemical analysis of breast cancer biopsies revealed that PML was overexpressed in a subset of breast cancers and enriched in triple-negative cases. Indeed, PML expression in breast cancer correlated strikingly with reduced time to recurrence, a gene signature of poor prognosis, and activated PPAR signaling. These findings have important therapeutic implications, as PML and its key role in fatty acid oxidation metabolism are amenable to pharmacological suppression, a potential future mode of cancer prevention and treatment.

In order for cancer cells to survive during metastasis, they must overcome anoikis, a caspase-dependent cell death process triggered by extracellular matrix (ECM) detachment, and rectify detachment-induced metabolic defects that compromise cell survival. However, the precise signals used by cancer cells to facilitate their survival during metastasis remain poorly understood. We have discovered that oncogenic Ras facilitates the survival of ECM-detached cancer cells by using distinct effector pathways to regulate metabolism and block anoikis. Surprisingly, we find that while Ras-mediated phosphatidylinositol (3)-kinase signaling is critical for rectifying ECM-detachment-induced metabolic deficiencies, the critical downstream effector is serum and glucocorticoid-regulated kinase-1 (SGK-1) rather than Akt. Our data also indicate that oncogenic Ras blocks anoikis by diminishing expression of the phosphatase PHLPP1 (PH Domain and Leucine-Rich Repeat Protein Phosphatase 1), which promotes anoikis through the activation of p38 MAPK. Thus, our study represents a novel paradigm whereby oncogene-initiated signal transduction can promote the survival of ECM-detached cells through divergent downstream effectors. Cell Death and Differentiation (2016) 23, 1271-1282 doi:10.1038/cdd.2016 published online 26 February 2016 Cancer metastasis, the spread of cancer cells to distant parts of the body, accounts for~90% of cancer-related deaths and represents an inherently difficult clinical challenge.1,2 It has become clear that for successful metastasis to occur, cells must overcome a caspase-dependent cell death mechanism, anoikis, which is triggered by detachment from the extracellular matrix (ECM).3 In addition to anoikis evasion, cancer cells must also contend with anoikis-independent cellular alterations that can compromise cellular viability. 4 Chief among these alterations are metabolic deficiencies that are induced by ECM detachment. [5][6][7] These metabolic alterations involve deficiencies in ATP generation, elevated levels of reactive oxygen species, and the induction of autophagy. 6,8,9 Although recent studies have begun to unravel the strategies used by cancer cells to ameliorate metabolic deficiencies during ECM detachment, 10 the signal-transduction cascades responsible for regulating metabolism during ECM detachment in cancer cells remain almost entirely unexplored.The activation of oncogenic signaling pathways is critical to anchorage-independent growth and ultimately to the survival of a variety of distinct cancer cell types during ECM detachment. 4,11 Presumably, this oncogenic signaling is also necessary for resolving the aforementioned ECM-detachment-induced metabolic deficiencies. ErbB2 overexpression in mammary epithelial cells results in a stimulation of phosphatidylinositol (3)-kinase (PI(3)K)/Akt signaling to promote glucose uptake and ATP generation.6 These data raise the question as to how cancer cells that lack ErbB2 overexpression rectify metabolic deficiencies during ECM detachment. Does activation o...

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Gephyronic acid is an antibiotic natural product isolated at the HZI (Helmholtz-Zentrum für Infektionsforschung) by Sasse and Höfle from the myxobacterium Archangium gephyra in 1995. Gephyronic acid has been shown to inhibit the growth of yeast and mold in the micromolar range; it also exhibits a cytostatic effect on mammalian cell cultures (human cervix, human leukemia, hamster kidney, hamster ovary, mouse connective tissue, and monkey kidney cancer cells) in the nanomolar range. An in vitro translation assay demonstrated that gephyronic acid is a specific inhibitor of eukaryotic protein synthesis. Interdisciplinary efforts within our laboratory are directed to the evaluation of gephyronic acid as a potential anticancer agent and its structural and biological relationship to the myriaporones and tedanolides. Our recent structural assignment and total synthesis of gephyronic acid incorporates a synthetic route that provides a means by which to access significant quantities of gephyronic acid efficiently and selectively, thus facilitating further biological evaluation. Additionally, we seek to identify the mode of action for gephyronic acid by screening various cancer cell lines through transcriptome and proteome profiling. Our proposed method will analyze mRNA expression levels of cells treated with gephyronic acid, or without for control, through the application of cDNA microarrays. Transcript profiling will then be coupled to proteomic profiling utilizing a method of protein labeling called iTRAQ. Identifying specific genes and their associated proteins will secure the mode of action of gephyronic acid selective activity and further defines potential as a chemotherapeutic lead. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C67.

Cancer cells utilize numerous signaling pathways to inhibit anoikis (detachment-induced apoptosis) and alter their metabolism to enable their survival in abnormal environments. Additional research on cancer cells’ ability to survive outside their normal niche has the potential to reveal mechanisms that may be targeted through the development of novel and more effective chemotherapeutics. One critical pathway that requires further understanding and has caught our interest is the Ras signaling pathway in mammary epithelial cells, and more specifically, the surprising role for SGK-1 in mediating cell death. We have engineered a mammary epithelial cell line (MCF-10A) that expresses constitutively active Ras (Ras G12V). To examine the ability of the Ras pathway to permit cell survival outside of the normal environment, we use an in vitro cell culture system that allows epithelial cells to grow in suspension and mimics the conditions when cells are detached from the extracellular matrix (ECM). Using this approach, we have found that the expression of Ras G12V causes cells to maintain their ATP levels when detached from the ECM and results in elevated levels of phosphorylated Akt. To further address the significance of the enhanced activation of Akt, we treated ECM-detached Ras G12V cells with varying dilutions of LY 294002, a PI(3)K inhibitor. As expected, we found that ATP levels decreased with elevated concentrations of this inhibitor, but surprisingly, phoshorylated Akt levels remained consistently high. This suggests the presence of a PI(3)K dependent but Akt independent signaling pathway that maintains ATP levels in detached cells expressing Ras G12V. We subsequently investigated the role of SGK-1, another kinase downstream of PI(3)K, as a possible player in the maintenance of ATP by Ras in detached cells. We found that treating suspended Ras G12V cells with GSK 650394, an inhibitor of SGK-1, decreased ATP levels in detached cells suggesting that SGK-1 is necessary for the Ras-mediated maintenance of ATP levels. To further investigate this result, we are using lentiviral-delivery of shRNA in our Ras G12V cells to obtain a cell line with stable knockdown of SGK-1. To continue to unveil a role for SGK-1 in Ras signaling, we are also making a constitutively active SGK-1 cell line in MCF-10A cells to examine the effects of active SGK-1 in the absence of Ras signaling. In summary, these data demonstrate an interesting role for SGK-1 in Ras-mediated maintenance of ATP levels when mammary epithelial cells are detached from the ECM. Our future research will continue to examine this important pathway to provide a better understanding of how cancer cells use the Ras pathway to survive outside their niche. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1238. doi:10.1158/1538-7445.AM2011-1238

In order for successful metastasis to occur, cells must overcome anoikis, a caspase-dependent cell death process triggered by detachment from the extracellular matrix (ECM). In addition, recent studies have revealed that ECM-detached cells must also rectify detachment-induced metabolic defects that compromise cell survival. However, the precise signals involved in the inhibition of anoikis and the restoration of proper cell metabolism during ECM-detachment are poorly understood. Of particular interest to our studies is the oncogene Ras, which is constitutively active in approximately 30% of all cancers and is well known to be regulate cell death pathways and metabolism. We have discovered that Ras facilitates the survival of ECM-detached cancer cells by utilizing distinct signaling pathways to block anoikis and regulate metabolism. Using MCF-10A cells engineered to overexpress oncogenic Ras and HCT116 cells (which contain an activating Ras mutation), we investigated the signaling pathways downstream of Ras that facilitate the survival of ECM-detached cells. Interestingly, we discovered that while Ras-mediated PI(3)K signaling is critical for rectifying metabolic defects during ECM-detachment, the downstream effector is not Akt, but rather SGK-1. SGK-1 stimulates glucose uptake, enhances ATP generation, promotes luminal filling in 3-dimensional cell culture, and drives anchorage-independent growth in soft agar. Interestingly, our data also indicate that oncogenic Ras utilizes an entirely distinct signaling pathway to block anoikis. We discovered that Ras diminishes the expression of the phosphatase PHLPP1. This inhibits the dephosphorylation-induced activation of a signaling cascade that culminates in the activation of pro-apoptotic p38 MAPK. In aggregate, these data unveil a novel survival strategy utilized by ECM-detached cancer cells and implicate both SGK-1 and PHLPP1 function downstream of Ras during ECM-detachment. The molecular mechanisms unveiled here could be utilized for the design of novel therapies that eliminate ECM-detached, metastatic cancer cells with Ras mutations through simultaneous modulation of SGK-1 and PHLPP1. Citation Format: Joshua A. Mason, Calli Versagli, Amy Leliaert, Sienna Durbin, Cassandra Buchheit, Zachary Schafer. Ras-mediated evasion of detachment-induced cell death involves differential signaling pathways for metabolism and anoikis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2148. doi:10.1158/1538-7445.AM2015-2148