Targeting eNOS in Pancreatic Cancer: A Long Road Ahead
Pancreatic ductal adenocarcinoma (PDAC) has the highest morbidity among cancers in the United States, posing a major challenge for therapeutic development. The potential role of endothelial nitric oxide synthase (eNOS/NOS3) in PDAC development and progression was implicated earlier. However, examination of eNOS as a potential therapeutic target by specific inhibitors in PDAC was not tested. In the present study, Lampson and colleagues examined the efficacy of a specific eNOS inhibitor, NG-nitro-l-arginine methyl ester (l-NAME), in relevant transgenic KrasG12D mutant (KC), KrasG12D/+ p53R172H (Trp53R172H/+) mutant (KPC) mice and in a human xenograft model (CFPac-1) of PDAC. The impact of genetic eNOS ablation (eNOS−/−/Nos3−/− mice) on PDAC development was also examined. They found high eNOS expression in both transgenic animals and human PDAC samples, essentially in the tumor vasculature. Genetic abrogation of eNOS markedly reduced the development of preinvasive pancreatic lesions. In the KC mice, significant reduction of the facial papilloma was achieved after l-NAME treatment. On the other hand, increased overall survival and tumor growth inhibition was observed in the KPC mice treated with l-NAME. In parallel, l-NAME treatment inhibited tumor growth of the human xenograft model of PDAC. Developing eNOS-based therapeutics could be useful for PDAC management, particularly when combined with other therapeutic approaches.
Mutant IDH1 Expression Results in Defects in Collagen Maturation and Basement Membrane Function in Brains of Transgenic Mice
Mutations in isocitrate dehydrogenase-1 (IDH1) occur in a substantial proportion of lower-grade gliomas (grade 2 and 3 diffuse glioma) and 5% to 10% of grade 4 gliomas (glioblastoma). Virtually all mutations occur at codon 132 and lead to a neomorphic enzyme, which results in increased production of 2-hydeoxyglutarate (2-HG). To understand the biology of IDH1 mutations in glioma, the investigators created a mouse knock-in of the IDH1R132H mutation, the most common mutation found in glioma. Mice with the floxed mutant Idh1 allele were crossed with Nestin-Cre transgenic mice. When driven by the nestin promoter, mice died in the perinatal period, with evidence of extensive brain hemorrhage. The authors then used a GFAP-Cre transgenic mouse, which showed similar findings, but of lesser severity. Approximately 8% of the mice survived until adulthood. Sasaki and colleagues examined the 2-HG metabolite and found it to be elevated in the Idh1-mutant knock-in mice. The level of 2-HG correlated positively with the presence of brain hemorrhage. The mutated IDH1 protein exhibits loss of catalytic activity for the normal α-ketoglutarate–NADPH–generating reaction and instead gains abnormal enzymatic activity that consumes NADPH. Therefore, the authors hypothesized that cells expressing the mutated IDH1 enzyme would show an increase in their intracellular NADP+/NADPH ratio. Extending this logic, because NADPH is a cofactor in the metabolism of antioxidant molecules, such as glutathione (GSH) and ascorbate, they reasoned that an increase in the NADP+/NADPH ratio would lead to a corresponding increase in intracellular reactive oxygen species (ROS). The authors did find an increase in the NADP+/NADPH in the knock-in mice but observed no change in ROS compared with controls. Instead, mutant IDH1 resulted in increased stability of hypoxia-inducible factor-1α (HIF1α), as well as its target genes, including Vegf. This result confirmed a prior finding that 2-HG blocked proline hydroxylation in HIF1α, accounting for increased stability. The authors then examined additional biologic processes in which prolyl and lysyl hydroxylases were involved, including the maturation of collagen, in which these enzymes play a key role. They found an increased overall level of collagen in the knock-in mice. However, the collagen present was immature due to an absence of function of prolyl and lysyl hydroxylases. Finally, the authors observed that mutant Idh1 knock-in mice did not develop glioma, and they speculated that additional alterations were required to generate tumors in this mouse model. Overall, the findings suggest that mutant IDH1 protein alters the brain microenvironment, leading to increased HIF1α and loss of collagen maturation. Elucidation of the ways in which these functions contribute to gliomagenesis will require further study.
Mechanistically Placing Alternative Splicing Downstream of RTK Signals
Zhou Z, Qiu J, Liu W, Zhou Y, Plocinik RM, Li H, et al. The Akt-SRPK-SR axis constitutes a major pathway in transducing EGF signaling to regulate alternative splicing in the nucleus. Mol Cell 2012;47:422–33.
Gene expression can be modulated exquisitely by alternative splicing at different sites on an mRNA to yield a wide variety of unique proteins. Signal transduction pathways, including the phosphoinositide 3-kinase (PI3K)/AKT pathway, are likely to play key roles in directing pre-mRNA splicing; however, mechanisms that underlie this level of regulatory control in the nucleus remain poorly understood. Zhou and colleagues have uncovered a novel signaling axis that links EGF signaling to regulation of alternative splicing. Their studies focused on serine/arginine–rich protein-specific kinases (SRPK), which specifically phosphorylate serine/arginine–rich splicing factors and were shown previously to be associated with cancers of the breast, colon, and pancreas. Human embryonic kidney 293 (HEK293) and HeLa cells transfected with the E1A splicing reporter minigene underwent a switch in splice-site selection upon EGF treatment, which resulted in production of the 13S isoform over the 9S isoform of E1A. This effect was abrogated in the presence of the PI3K inhibitor Wortmannin and was unaffected by a protein kinase C inhibitor, suggesting that the PI3K/AKT signaling pathway is important for EGF-induced alternative splicing. Cells with constitutively active AKT, SRPK1, or SRPK2 overexpression produced the same splicing variant as cells treated with EGF, whereas the kinase dead AKT or SRPK1/2 mutants displayed the same impairment in splice-site switching as cells treated with Wortmannin. siRNA knockdown of either SRPK1 or SRPK2 was sufficient to prevent EGF-induced alternative splicing, even in the presence of AKT activation, which suggests that these 2 kinases have nonredundant functions.
Zhou and colleagues used a pharmacologic approach to map the position of SRPKs in the EGF signal cascade. In contrast with the effects observed upon PI3K inhibition, they found that treatment of cells overexpressing SRPK1/2 with mTOR inhibitors did not perturb the E1A splice-site switching. Therefore, it is likely that SRPKs are positioned between PI3K and mTOR in the EGF pathway. An RNA annealing, selection, and ligation assay with high-throughput sequencing (RASL-seq) in a panel of 3,726 evolutionarily conserved alternative splicing events revealed a large subset of samples that undergo splice-site switching upon EGF treatment. Importantly, this global effect on alternative splicing was abolished upon SRPK knockdown by RNA interference (RNAi), further supporting a critical role for SRPK in EGF-mediated alternative splicing. Systematic pharmacologic inhibition of major EGF-activated signal transduction pathways (including JAK/STAT, extracellular signal-regulated kinase, PI3K/AKT, and mTOR) showed that the PI3K pathway is most critical for the EGF-directed splicing program. Mass spectrometry analysis of phosphopeptides from immunoprecipitated SRPK1 uncovered multiple phosphorylation sites, and in vitro kinase assays showed that AKT promotes SRPK autophosphorylation. The authors identified 2 putative SRPK1 autophosphorylation sites that, when mutated to alanine, prevented an EGF-induced E1A splice-site switch. When these autophosphorylation sites were mutated to aspartic acid, Wortmannin did not inhibit alternative splicing. Nuclear translocation of SRPK1/2 and serine-argonine–rich splicing factor phosphorylation were observed in EGF-treated cells, but these activities were inhibited upon Wortmannin administration. The authors used coimmunoprecipitation experiments to determine that SRPK1/2 switches from associating with HSP70 to HSP90 in the presence of EGF to aid translocation of SRPK to the nucleus. In addition, they show that the 14-3-3 family of proteins acts to prevent excessive accumulation of HSPs in the nucleus.
These data suggest that HSPs and 14-3-3 proteins may coordinate SRPK activity upon EGF stimulation. Overall, these findings reveal a novel signaling axis in which EGF signaling activates PI3K/AKT, which initiates SRPK autophosphorylation and modulates the association of this protein with its molecular chaperones. This coordinated activity ultimately directs SRPK translocation to the nucleus to regulate pre-mRNA alternative splicing. SRPKs have previously been associated with a variety of cancers, and this new regulatory pathway may represent a new and druggable node in tumorigenesis.
An Aspirin a Day May Keep Luminal Breast Cancer at Bay
Kuznetsov HS, Marsh T, Markens BA, Castaño Z, Greene-Colozzi A, Hay SA, et al. Identification of luminal breast cancers that establish a tumor supportive macroenvironment defined by proangiogenic platelets and bone marrow derived cells. Cancer Discov; Published OnlineFirst August 15, 2012; doi:10.1158/2159-8290.CD-12-0216.
Although breast tumors can disseminate before surgery, we do not fully understand how and why they develop into clinically significant metastases. Kuznetsov and colleagues have defined a novel mechanism by which distant tumors may stimulate progression of indolent breast cancer metastases. Malignant cells of luminal breast cancer (LBC) or triple-negative breast cancer (TNBC) that were grown in nude mice were shown to stimulate the growth of a usually indolent breast cancer cell line injected into the opposite flank. TNBC-instigated tumors were infiltrated by a desmoplastic stroma of myofibroblasts, whereas the LBC-instigated tumors were extensively vascularized with VEGF receptor 2+ (VEGFR2+) blood vessels. Further experiments showed that the LBC instigators acted systemically, causing bone marrow expansion of endothelial progenitors that were then mobilized to the responding tumors. This raised the question of how the LBC instigator tumor was able to have these systemic effects. Although there were no obvious circulating cytokine candidates, a significant increase in platelet counts was observed in the mice bearing LBC-instigator tumors. Moreover, the responding tumor cells had high levels of surface CD24, which can bind P-selectin located on platelets. The blood vessels in the responding tumor had areas of exposed collagen, a known stimulus for platelet recruitment. Platelets were recruited to these tumors, and activated platelets from mice bearing LBC-instigator tumors exhibited angiogenic activity. In further experiments the authors found that platelets packaged proangiogenic and proinflammatory factors produced by the LBC-instigator tumors. A human primary LBC biopsy implanted in the same manner was also able to stimulate CD24 enrichment, platelet recruitment, and VEGFR2+ cell recruitment in the usually indolent responder tumors, and LBC-instigator tumors stimulated primary human breast and renal cancer organoids to form vascularized tumors in the opposite flank. The importance of platelets in this systemic stimulation of breast tumor growth was highlighted in mice treated with aspirin, a known inhibitor of platelet activity. Aspirin reduced LBC-instigated tumor formation and completely inhibited recruitment of VEGFR2+ cells to any tumors that did develop. These findings are especially interesting in view of emerging epidemiologic data indicating that daily aspirin can reduce death from adenocarcinoma, particularly in patients without detectable metastasis at diagnosis.
Note: Breaking Advances are written by Cancer Research Editors. Readers are encouraged to consult the articles referred to in each item for full details on the findings described.
- ©2012 American Association for Cancer Research.