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The M2 pyruvate kinase (PKM2) isoform is upregulated in most cancers and plays a crucial role in regulation of the Warburg effect, which is characterized by the preference for aerobic glycolysis over oxidative phosphorylation for energy metabolism. PKM2 is an alternative-splice isoform of the PKM gene and is a potential therapeutic target. Antisense oligonucleotides (ASO) that switch PKM splicing from the cancer-associated PKM2 to the PKM1 isoform have been shown to induce apoptosis in cultured glioblastoma cells when delivered by lipofection. Here, we explore the potential of ASO-based PKM splice switching as a targeted therapy for liver cancer. A more potent lead constrained-ethyl (cEt)/DNA ASO induced PKM splice switching and inhibited the growth of cultured hepatocellular carcinoma (HCC) cells. This PKM isoform switch increased pyruvate-kinase activity and altered glucose metabolism. In an orthotopic HCC xenograft mouse model, the lead ASO and a second ASO targeting a nonoverlapping site inhibited tumor growth. Finally, in a genetic HCC mouse model, a surrogate mouse-specific ASO induced Pkm splice switching and inhibited tumorigenesis, without observable toxicity. These results lay the groundwork for a potential ASO-based splicing therapy for HCC. Significance: Antisense oligonucleotides are used to induce a change in PKM isoform usage in hepatocellular carcinoma, reversing the Warburg effect and inhibiting tumorigenesis.

KRAS is recurrently mutated in pancreatic ductal adenocarcinoma (PDAC), triggering the formation of acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN). However, the majority of pancreatic cells from KC (LSL-KrasG12D/+; Pdx-1-Cre) mice carrying the KrasG12D mutation remain morphologically normal for a long time, suggesting the existence of compensatory feedback mechanisms that buffer KrasG12D signaling, and that additional steps are required for disrupting cell homeostasis and promoting transformation. Here we found that splicing factor SRSF1, which is associated with cell transformation, is downregulated in the majority of morphologically normal pancreas cells with the KrasG12D mutation as a compensatory feedback mechanism. To assess the role of SRSF1 in PDAC, we generated a transgenic mouse strain (SC) with Dox-inducible pancreatic-specific expression of SRSF1 protein. Elevated SRSF1 alone is sufficient to induce pancreatitis and ADM transformation. By further crossing the SC strain with KrasG12D alone (KSC) or together with Trp53R172H (KPSC), we found that increasing SRSF1 accelerated KrasG12D-mediated tumor initiation and resulted in more aggressive PDAC. To address the underlying mechanisms of SRSF1’s involvement in the homeostatic response to KrasG12D mutation and in PDAC initiation, we generated organoid lines from the above mouse strains. Following Dox induction, elevated SRSF1 with Kras WT or KrasG12D consistently activated MAPK signaling, which is one of the top negatively enriched pathways in response to KrasG12D. Furthermore, SRSF1 promoted the expression of Il1r1—an IL1 receptor associated with the activation of MAPK signaling—by regulating an alternative-splicing event in the 5’UTR to generate a more stable mRNA isoform. We conclude that decreased SRSF1 is a compensatory feedback mechanism in pancreatic cells against the KrasG12D mutation, whose disruption facilitates PDAC initiation. Citation Format: Ledong Wan, Kuan-Ting Lin, Mohammad A. Rahman, Zhikai Wang, Youngkyu Park, David A. Tuveson, Adrian R. Krainer. Loss of compensatory feedback mechanism involving splicing factor SRSF1 accelerates KrasG12D-mediated pancreatic cancer initiation [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-011.

Cancer cells often display aberrant profiles of alternative splicing, leading to the production of isoforms that can stimulate cell proliferation and migration, increase resistance to apoptosis, or alter cell metabolism. Recently, recurrent somatic mutations in components of the splicing machinery have been identified in human tumors, raising a new interest in the field and suggesting that alterations in splicing factors are a new hallmark of cancer. Splicing factors elicit changes in splicing in a concentration-dependent manner. Thus, changes in the expression of these proteins, as reported in different types of cancers, can affect the splicing of multiple genes and are likely involved in splicing deregulation in cancer, even in the absence of mutations. We previously demonstrated that the splicing factor SRSF1'formerly SF2/ASF' is often upregulated in human breast cancers and can transform mammary epithelial cells in vivo and in vitro. SRSF1 is a prototypical member of the SR protein family, composed of 12 members sharing structural similarities. However, little is known about differences and redundancies in their splicing targets and thus in their specific biological functions. We are now investigating wherever additional splicing factors can promote mammary epithelial cell transformation by using models that mimic the correct biological context in which tumors arise. We have mined data from a large collection of human breast tumors and cancer cell lines to identify splicing factors overexpressed at the transcript and protein levels. We then compared the ability of 10 selected splicing factors to transform human mammary epithelial MCF-10A cells grown in organotypic 3-D culture. Under these conditions, the cells undergo a 16-day differentiation program, forming growth-arrested, hollow acinar structures with polarized architecture, similar to the terminal units of mammary ducts. Various oncogenes associated with breast cancer are known to disrupt acinar growth and/or architecture. We assessed how splicing-factor overexpression affects cell proliferation and apoptosis, as well as cell migration and invasion. Interestingly, only certain splicing factors are oncogenic in this context, suggesting functional differences. Furthermore, specific splicing factors increase cell migration and invasion but are unable to promote transformation. We have demonstrated specificity in splicing-factor mediated transformation, and we are now investigating what are the relevant splicing targets for transformation by several of these oncogenic splicing factors. Furthermore, we are investigating which splicing factors are necessary or sufficient to promote cell invasion and metastatic potential in human breast cancer cell lines in vitro and in vivo. Finally, we have identified splicing factors that cooperate specifically with the MYC oncogene and are significantly co-expressed with MYC in human breast tumors. By identifying oncogenic splicing factors that can promote breast cancer, and their downstream effectors, we hope to establish candidate targets for therapeutics development based on the modulation of oncogenic splicing events and their regulators. Citation Format: Olga Anczukow, Kuan-Ting Lin, Martin Akerman, Shipra Das, Senthil K. Muthuswamy, Adrian Krainer. Differential functions of splicing factors in breast-cancer initiation and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B53.

Multiple dys-regulated cell signaling pathways and extensive cross-talks, which can be depicted via protein-protein interactions (PPIs), among individual signaling pathways, characterize cancer development and progression. However, systematic analysis of the cross-talk events from distinct pathways via PPIs remains fragmentary, and the reliability of PPI datasets, particularly at the cellular level, remains to be vigorously examined. Here, we first build the cross-talk pathway map of heptocellular carcinoma (HCC) through overexpression patterns in HCC among 23 pathways. By detecting endogenous PPIs in two HCC cell lines, Huh7 (low invasive cells) and Mahlavu (high invasive cells), we validate 50 PPI events within pathway and 19 PPI-mediated cross-talk events among 23 pathways in HCC. Not surprisingly, many distinct PPIs are present between Huh7 and Mahlavu, providing the opportunity to characterize tumor invasion. By analyzing topology of cross-talk PPI network, CRKL has a pivotal role in cross-talk pathway. This initial attempt to map the cross-talk pathways in HCC may represent the first step in deciphering hepatocarcinogenesis at the systems level and might reveal a novel strategy for HCC treatment. 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 4929. doi:10.1158/1538-7445.AM2011-4929

Signal transduction pathways at cellular level require massive protein-protein interactions (PPIs) in response to different environmental cues. Diverse experimental techniques for detecting PPIs have been developed; however, the large amount of data accumulated from various sources has posed a grand challenge in data reliability for PPI. To evaluate the role of PPIs within signaling pathways, we have systematic profiled PPI residing in 28 KEGG pathways, for example mTOR, MAPK, Wnt, VEGF, and Hedgehog, and 3 cancer-related pathways stored in KEGG (http://www.genome.jp/kegg/). Of ∼60000 human PPI collected, 2786 PPI can be detected within targeted 14 pathways. To validate these PPIs, we used a newly developed Proximity Ligation Assay, which detects endogenous PPI through a pair of antibodies that bind to proteins in close proximity. Each PPI can be made visible through DNA amplification and quantified by counting fluorescent dots in the cell. Of 1,204 PPIs examined, 557 PPIs can be confirmed in HeLa cells. Specifically, 383 out of 557 validated PPIs residing within pathways in cancer and mTOR, Wnt and MAPK pathways are considered novel “links” based on annotations of KEGG. Moreover, 79 validated PPIs are cross-talk events among 35 signaling transduction and cancer related pathways. Surprisingly, only 16 PPIs (out of 557 confirmed PPIs) are highly correlated in 7 sets of cancer cells from NCI60 microarray dataset. In this large scale PPI profiling, 41 apoptosis related PPIs were further tested in 3 cancer cell lines to explore the possibility for drug screening. These systematic analyses of endogenous PPI pairs within and between pathways may set the foundation to disrupt the signaling pathways by targeting PPIs in cancer drug discovery. 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 1976. doi:10.1158/1538-7445.AM2011-1976