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IL-1b neutralization sensitizes PDA tumors to PD-1 checkpoint blockade.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy typified by a highly stromal and weakly immunogenic tumor microenvironment that promotes tumor evolution and contributes to therapeutic resistance. Here, we demonstrate that PDA tumor cell–derived proinflammatory cytokine IL1β is essential for the establishment of the protumorigenic PDA microenvironment. Tumor cell–derived IL1β promoted the activation and secretory phenotype of quiescent pancreatic stellate cells and established an immunosuppressive milieu mediated by M2 macrophages, myeloid-derived suppressor cells, CD1dhiCD5+ regulatory B cells, and Th17 cells. Loss of tumor cell–derived IL1 signaling in tumor stroma enabled intratumoral infiltration and activation of CD8+ cytotoxic T cells, attenuated growth of pancreatic neoplasia, and conferred survival advantage to PDA-bearing mice. Accordingly, antibody-mediated neutralization of IL1β significantly enhanced the antitumor activity of α-PD-1 and was accompanied by increased tumor infiltration of CD8+ T cells. Tumor cell expression of IL1β in vivo was driven by microbial-dependent activation of toll-like receptor 4 (TLR4) signaling and subsequent engagement of the NLRP3 inflammasome. Collectively, these findings identify a hitherto unappreciated role for tumor cell–derived IL1β in orchestrating an immune-modulatory program that supports pancreatic tumorigenesis.Significance:These findings identify a new modality for immune evasion in PDA that depends on IL1β production by tumor cells through TLR4-NLRP3 inflammasome activation. Targeting this axis might provide an effective PDA therapeutic strategy.

Universal CAR T-cell therapies are poised to revolutionize cancer treatment and to improve patient outcomes. However, realizing these advantages in an allogeneic setting requires universal CAR T-cells that can kill target tumor cells, avoid depletion by the host immune system, and proliferate without attacking host tissues. Here, we describe the development of a novel immune-evasive CAR T-cells scaffold that evades NK cell and alloresponsive T-cell attacks and imparts efficient antitumor activityin vitroandin vivo. This scaffold could enable the broad use of universal CAR T-cells in allogeneic settings and holds great promise for future powerful clinical applications.

Summary: Misregulation of alternative splicing is a hallmark of human tumors, yet to what extent and how it contributes to malignancy are only beginning to be unraveled. Here, we define which members of the splicing factor SR and SR-like families contribute to breast cancer and uncover differences and redundancies in their targets and biological functions. We identify splicing factors frequently altered in human breast tumors and assay their oncogenic functions using breast organoid models. We demonstrate that not all splicing factors affect mammary tumorigenesis in MCF-10A cells. Specifically, the upregulation of SRSF4, SRSF6, or TRA2β disrupts acinar morphogenesis and promotes cell proliferation and invasion in MCF-10A cells. By characterizing the targets of these oncogenic splicing factors, we identify shared spliced isoforms associated with well-established cancer hallmarks. Finally, we demonstrate that TRA2β is regulated by the MYC oncogene, plays a role in metastasis maintenance in vivo, and its levels correlate with breast cancer patient survival. : Park et al. demonstrate that >50% of human breast tumors exhibit an alteration in one of the splicing factors from the SR protein family. Using in vitro and in vivo breast cancer models, they identify three splicing factors that promote cell proliferation and invasion by regulating isoforms associated with cancer hallmarks. Keywords: alternative RNA splicing, breast cancer, splicing factor, SR protein, metastasis, MYC, TRA2-beta, triple negative breast cancer

The immune microenvironment of pancreatic ductal adenocarcinoma (PDAC) is comprised of a heterogeneous population of cells that are critical for disease evolution. Prominent among these are the specialized CD1dhiCD5+ regulatory B (Breg) cells that exert a pro-tumorigenic role by promoting tumor cell proliferation. Dissecting the molecular pathways regulating this immune sub-population can thus be valuable for uncovering potential therapeutic targets. Here, we investigate Bruton’s Tyrosine Kinase (BTK), a key B cell kinase, as a potential regulator of CD1dhiCD5+ Breg differentiation in the pancreatic tumor microenvironment. Treatment of cytokine-induced B cells in vitro with the high specificity BTK inhibitor Tirabrutinib inhibited CD1dhiCD5+ Breg differentiation and production of IL-10 and IL-35, essential mediators of Breg immunosuppressive functions. The BTK signaling pathway was also found to be active in vivo in PanIN-associated regulatory B cells. Tirabrutinib treatment of mice bearing orthotopic KrasG12D-pancreatic lesions severely compromised stromal accumulation of the CD1dhiCD5+ Breg population. This was accompanied by an increase in stromal CD8+IFNγ+ cytotoxic T cells and significant attenuation of tumor cell proliferation and PanIN growth. Our results uncover a novel role for BTK in regulating CD1dhiCD5+ Breg differentiation and emphasize its potential as a therapeutic target for pancreatic cancer.

International audience; Host versus graft reaction is a major impediment to CAR-T cell immune therapy in allogeneic settings. Authors show here that CAR-T cells, engineered to be deficient in MHC I expression but to express the NK inhibitor HLA-E, are resistant to destruction by both T and NK cells of the host. Universal CAR T-cell therapies are poised to revolutionize cancer treatment and to improve patient outcomes. However, realizing these advantages in an allogeneic setting requires universal CAR T-cells that can kill target tumor cells, avoid depletion by the host immune system, and proliferate without attacking host tissues. Here, we describe the development of a novel immune-evasive universal CAR T-cells scaffold using precise TALEN-mediated gene editing and DNA matrices vectorized by recombinant adeno-associated virus 6. We simultaneously disrupt and repurpose the endogenous TRAC and B2M loci to generate TCR alpha beta- and HLA-ABC-deficient T-cells expressing the CAR construct and the NK-inhibitor named HLA-E. This highly efficient gene editing process enables the engineered T-cells to evade NK cell and alloresponsive T-cell attacks and extend their persistence and antitumor activity in the presence of cytotoxic levels of NK cell in vivo and in vitro, respectively. This scaffold could enable the broad use of universal CAR T-cells in allogeneic settings and holds great promise for clinical applications.

Background: Several studies support the influence of ethnicity on the relationship between body mass index (BMI) and risk factors for cardiovascular disease. Aim and Objectives: This study aims to investigate the difference in correlation between obesity profile and blood pressure (BP) between Sikkimese and immigrant students in Gangtok. Materials and Methods: A cross-sectional study was conducted in the Department of Physiology, Sikkim Manipal Institute of Medical Sciences, Sikkim, Gangtok. A total of 274 students aged 18–25 years were included in the study. The anthropometric parameters (Height, weight, BMI, and Waist Circumference [WC]), systolic and diastolic BP (DBP) of the ethnic and immigrant group, were recorded. Two-tail Students’ test was performed to compare two groups and Pearson product-moment correlation coefficient was used to measure the linear correlation. Results: Mean value of all variables-BMI, WC, Systolic BP (SBP), and DBP was higher in the immigrant group as compared to the ethnic group, but the differences were not statistically significant. Lower prevalence of general obesity, abdominal obesity, elevated BP., and hypertension was found among Sikkimese compared to non-Sikkimese young adults. A stronger positive correlation was found between BP and WC and between BP and BMI in immigrants compared to the ethnic group. Conclusion: Higher incidence of different cardiovascular risk factors in young immigrant adults as compared to the ethnic Sikkimese group in this study points toward ethnicity-dependent differences in the relationship between obesity profile and BP.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy typified by a highly stromal and weakly immunogenic tumor microenvironment that promotes tumor evolution and contributes to therapeutic resistance. Here, we demonstrate that PDA tumor cell–derived proinflammatory cytokine IL1β is essential for the establishment of the protumorigenic PDA microenvironment. Tumor cell–derived IL1β promoted the activation and secretory phenotype of quiescent pancreatic stellate cells and established an immunosuppressive milieu mediated by M2 macrophages, myeloid-derived suppressor cells, CD1dhiCD5+ regulatory B cells, and Th17 cells. Loss of tumor cell–derived IL1 signaling in tumor stroma enabled intratumoral infiltration and activation of CD8+ cytotoxic T cells, attenuated growth of pancreatic neoplasia, and conferred survival advantage to PDA-bearing mice. Accordingly, antibody-mediated neutralization of IL1β significantly enhanced the antitumor activity of α-PD-1 and was accompanied by increased tumor infiltration of CD8+ T cells. Tumor cell expression of IL1β in vivo was driven by microbial-dependent activation of toll-like receptor 4 (TLR4) signaling and subsequent engagement of the NLRP3 inflammasome. Collectively, these findings identify a hitherto unappreciated role for tumor cell–derived IL1β in orchestrating an immune-modulatory program that supports pancreatic tumorigenesis. Significance: These findings identify a new modality for immune evasion in PDA that depends on IL1β production by tumor cells through TLR4-NLRP3 inflammasome activation. Targeting this axis might provide an effective PDA therapeutic strategy.

SUMMARYMisregulation of alternative splicing is a hallmark of human tumors; yet to what extent and how it contributes to malignancy are only beginning to be unraveled. Here, we define which members of the splicing factor SR and SR-like families contribute to breast cancer, and uncover differences and redundancies in their targets and biological functions. We first identify splicing factors frequently altered in human breast tumors, and then assay their oncogenic functions using breast organoid models. Importantly we demonstrate that not all splicing factors affect mammary tumorigenesis. Specifically, upregulation of either SRSF4, SRSF6 or TRA2β promotes cell transformation and invasion. By characterizing the targets of theses oncogenic factors, we identify a shared set of spliced genes associated with well-established cancer hallmarks. Finally, we demonstrate that the splicing factor TRA2β is regulated by the MYC oncogene, plays a role in metastasis maintenancein vivo, and its levels correlate with breast-cancer-patient survival.

Pancreatic intraepithelial neoplasms (PanINs) are well-defined precursor lesions of pancreatic ductal adenocarcinoma (PDA). The initiation and progression of PanINs is marked by dynamic stromal reconstruction and localized acute inflammation. Using an orthotopic transplantation model of early and advanced pancreatic neoplasia in immunocompetent recipient mice, we have uncovered a crucial role for the cytokine IL-1b in KRasG12D-driven PanIN evolution. Tumor cell-derived IL-1b¢ was found to be critical for shaping the protumorigenic PanIN microenvironment by promoting activation of quiescent pancreatic stellate cells and establishing an immunosuppressive milieu mediated by M2 macrophages, MDSCs and CD1dhiCD5+ regulatory B cells. Furthermore, loss of IL-1 signaling in tumor stroma activates the cytotoxic T-cell immune response, significantly attenuates PanIN growth and confers survival advantage to PDAC-bearing mice. Tumor cell expression of IL-1b in vivo is driven by upregulation of TLR4 signaling and activation of the NLRP3 inflammasome. KrasG12D enhances surface TLR4 expression, sensitizing transformed cells to TLR4 ligands in the microenvironment, including the pancreatic microbiome. Collectively, our study describes a complex tumor-stromal interplay orchestrated by the TLR4-NLRP3-IL-1b signaling axis, which is critical for shaping the tumor microenvironment and driving pancreatic tumorigenesis. Citation Format: Shipra Das, Sandra Vogt, Christina Hadju, Dafna Bar-Sagi. Regulation and function of IL-1b in immune modulation of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B39.

With the recent breakthroughs in immunotherapy as curative treatments in certain tumor types, there has been renewed interest in the relationship between immunity and tumor growth. Although we are gaining a greater understanding of the complex interplay of immune modulating components in the tumor microenvironment, the specific role that tumor cells play in shaping the immune milieu is still not well characterized. In this review, we focus on how mutant Kras tumor cells contribute to tumor immunity, with a specific focus on processes induced directly or indirectly by the oncogene.

Splicing and translation are highly regulated steps of gene expression. Altered expression of proteins involved in these processes can be deleterious. Therefore, the cell has many safeguards against such misregulation. We report that the oncogenic splicing factor SRSF1, which is overexpressed in many cancers, stabilizes the tumor suppressor protein p53 by abrogating its MDM2-dependent proteasomal degradation. We show that SRSF1 is a necessary component of an MDM2/ribosomal protein complex, separate from the ribosome, that functions in a p53-dependent ribosomal-stress checkpoint pathway. Consistent with the stabilization of p53, increased SRSF1 expression in primary human fibroblasts decreases cellular proliferation and ultimately triggers oncogene-induced senescence (OIS). These findings underscore the deleterious outcome of SRSF1 overexpression and identify a cellular defense mechanism against its aberrant function. Furthermore, they implicate the RPL5-MDM2 complex in OIS and demonstrate a link between spliceosomal and ribosomal components, functioning independently of their canonical roles, to monitor cellular physiology and cell-cycle progression.

SummaryThe SR protein splicing factor SRSF1 is a potent proto-oncogene that is frequently upregulated in cancer. Here, we show that SRSF1 is a direct target of the transcription factor oncoprotein MYC. These two oncogenes are significantly coexpressed in lung carcinomas, and MYC knockdown downregulates SRSF1 expression in lung-cancer cell lines. MYC directly activates transcription of SRSF1 through two noncanonical E-boxes in its promoter. The resulting increase in SRSF1 protein is sufficient to modulate alternative splicing of a subset of transcripts. In particular, MYC induction leads to SRSF1-mediated alternative splicing of the signaling kinase MKNK2 and the transcription factor TEAD1. SRSF1 knockdown reduces MYC's oncogenic activity, decreasing proliferation and anchorage-independent growth. These results suggest a mechanism for SRSF1 upregulation in tumors with elevated MYC and identify SRSF1 as a critical MYC target that contributes to its oncogenic potential by enabling MYC to regulate the expression of specific protein isoforms through alternative splicing.

A salient feature of pancreatic ductal adenocarcinoma (PDAC) is an abundant fibroinflammatory response characterized by the recruitment of immune and mesenchymal cells and the consequent establishment of a protumorigenic microenvironment. Here, we report the prominent presence of B cells in human pancreatic intraepithelial neoplasia and PDAC lesions as well as in oncogenic Kras-driven pancreatic neoplasms in the mouse. The growth of orthotopic pancreatic neoplasms harboring oncogenic Kras was significantly compromised in B-cell–deficient mice (μMT), and this growth deficiency could be rescued by the reconstitution of a CD1dhiCD5+ B-cell subset. The protumorigenic effect of B cells was mediated by their expression of IL35 through a mechanism involving IL35-mediated stimulation of tumor cell proliferation. Our results identify a previously unrecognized role for IL35-producing CD1dhiCD5+ B cells in the pathogenesis of pancreatic cancer and underscore the potential significance of a B-cell/IL35 axis as a therapeutic target. Significance: This study identifies a B-cell subpopulation that accumulates in the pancreatic parenchyma during early neoplasia and is required to support tumor cell growth. Our findings provide a rationale for exploring B-cell–based targeting approaches for the treatment of pancreatic cancer. Cancer Discov; 6(3); 247–55. ©2015 AACR. See related commentary by Roghanian et al., p. 230. See related article by Lee et al., p. 256. See related article by Gunderson et al., p. 270. This article is highlighted in the In This Issue feature, p. 217

Biotechnological applications of algal polysaccharide as emulsifiers, thickeners and laxatives have led to the screening and selection of certain diazotrophic filamentous cyanobacteria from saline/alkaline soil of Madhya Pradesh, India. Strain specific variation in cell bound, extracellular and total polysaccharide content was quantified under laboratory conditions. Among the cyanobacterial isolates examined Nostoc calcicola RDU-3 was found to produce highest amount (105 mg l−1) of extracellular polysaccharide on 44th day of growth under diazotrophic growth conditions. Extracellular polysaccharide production of cyanobacterium Nostoc calcicola RDU-3 was optimal at pH-10, temperature 35°C, photoperiod of 24 h and in white light. The Gas Chromatographic analysis of polysaccharide from Nostoc calcicola RDU-3 revealed the presence of ribose (36.03%), xylose (34.13%), rhamnose (29.67%) and glucose (4.0%). The polysaccharide is novel in that it possesses ribose as the predominant monosaccharide with very low levels of glucose. Predominance of ribose monosaccharide is the unique feature which is reported to be used as metabolic supplement to the heart. IR spectrum of extracellular polysaccharide revealed the presence of sulphate group. Such sulphated polysaccharide is reported to have antiviral properties.

Background: Overweight, obesity, and metabolic syndrome (MetS) are rapidly increasing in India. Aims and Objectives: This study demonstrated the prevalence of generalized, abdominal obesity including intra-abdominal and subcutaneous adiposity along with other associated factors in young adults. Materials and Methods: A cross-sectional study was conducted with 200 subjects. The anthropometric parameters (body mass index [BMI], waist circumference [WC], and skinfold thickness), fasting blood glucose (FBG), and blood pressure were recorded. Percentage of body fat (BF), total abdominal fat (TAF), intra-abdominal adipose tissue (IAAT), subcutaneous adipose tissue (SCAT), and basal metabolic rate (BMR) were measured by predictive equations. Data were analyzed using t-test, analysis of variance, and Pearson’s correlation tests. P < 0.05 was considered statistically significant. Results: The prevalence of generalized obesity (GO) (by BMI [>25 kg/m2]) was 11%. The prevalence of abdominal obesity according to WC was 17 %, whereas that measured by TAF was 8%. Increased IAAT was more in females (26.02%) as compared to males (8%) with overall prevalence 16.5%. The overall prevalence of SCAT was 27%, more in males (41.56%) as compared to females (17.89%). The prevalence of impaired FBG was 19% (prediabetic), MetS 5.5%, hypertension according to systolic blood pressure 6%, and according to diastolic blood pressure 13%. The predictive BMR was significantly higher with obese subjects as compared to healthy members in both sexes (P < 0.05). Conclusion: The prevalence of GO, abdominal obesity, regional adiposity, and MetS among young adults necessitates public health intervention.

Background During spliceosome assembly, protein-protein interactions (PPI) are sequentially formed and disrupted to accommodate the spatial requirements of pre-mRNA substrate recognition and catalysis. Splicing activators and repressors, such as SR proteins and hnRNPs, modulate spliceosome assembly and regulate alternative splicing. However, it remains unclear how they differentially interact with the core spliceosome to perform their functions. Results Here, we investigate the protein connectivity of SR and hnRNP proteins to the core spliceosome using probabilistic network reconstruction based on the integration of interactome and gene expression data. We validate our model by immunoprecipitation and mass spectrometry of the prototypical splicing factors SRSF1 and hnRNPA1. Network analysis reveals that a factor’s properties as an activator or repressor can be predicted from its overall connectivity to the rest of the spliceosome. In addition, we discover and experimentally validate PPIs between the oncoprotein SRSF1 and members of the anti-tumor drug target SF3 complex. Our findings suggest that activators promote the formation of PPIs between spliceosomal sub-complexes, whereas repressors mostly operate through protein-RNA interactions. Conclusions This study demonstrates that combining in-silico modeling with biochemistry can significantly advance the understanding of structure and function relationships in the human spliceosome. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0682-5) contains supplementary material, which is available to authorized users.

Oncogene-induced senescence (OIS) is a protective mechanism through which normal cells defend themselves against transformation.1 Oncogenic stress (induced by mutation or proto-oncogene overexpression) activates this tumor-suppressive pathway, wherein cells enter a stage of irreversible cell-cycle arrest, thereby restricting uncontrolled cellular proliferation and malignant growth. OIS has been described both in vitro in primary cells, as well as in vivo in pre-malignant lesions, for multiple oncogenes and in various cellular contexts. We recently reported a unique form of OIS activated by overexpression of the oncogenic splicing factor SRSF1.2 SRSF1 encodes a multi-functional protein with regulatory roles in many aspects of RNA biogenesis and function, including constitutive and alternative splicing, mRNA export and translation.3 Aberrant SRSF1 activity is deleterious for the cell: SRSF1 depletion triggers genomic instability, cell-cycle arrest and apoptosis,4 whereas its overexpression leads to oncogenic transformation.5 Consequently, SRSF1 expression is strictly regulated. SRSF1 autoregulates its expression through multiple post-transcriptional and translational mechanisms.3 Our recent finding that SRSF1 keeps a check on the oncogenic outcome of its overexpression by activating a novel OIS pathway represents yet another mechanism of SRSF1 autoregulation. Interestingly, senescence induction by SRSF1 is tightly coupled to the ribosomal-stress-response pathway, which was previously shown to stabilize the critical cell-cycle regulator and tumor-suppressor protein p53, upon ribosomal perturbation.6 Ribosome biogenesis and function are critical regulators of cell growth and proliferation, and are highly sensitive to nutrient and growth-factor availability, as well as oncogenic burden. Aberrant ribosome assembly or function triggers formation of a complex of ribosomal proteins, including RPL5 and RPL11, with the E3-ubiquitin ligase MDM2. Sequestration of MDM2 in this nucleoplasmic complex inhibits ubiquitylation of the primary MDM2 substrate, p53, promoting its stability. We demonstrated that SRSF1 interacts with both RPL5 and MDM2, and this interaction is promoted by inducers of ribosomal stress, indicating a role of SRSF1 in the ribosomal-stress pathway. Consistent with the established RPL5-MDM2 function, SRSF1 overexpression decreases p53’s ubiquitylation and increases its stability at the protein level, without affecting TP53 transcription, mRNA splicing or mRNA stability. Furthermore, upon overexpression in primary human and murine cells, SRSF1 limits its own oncogenic activity by recruiting the RPL5-MDM2 complex to promptly activate a tumor-suppressive barrier, i.e., p53-mediated premature cellular senescence. Our results provide new insights into the mechanisms of both ribosomal stress and OIS. Previous reports on the RP-MDM2 complex described ternary and quaternary complexes comprising RPL5, MDM2 and other ribosomal proteins, primarily RPL11 and RPL23.6 The RPL5-MDM2 interaction was reported to be strengthened in the presence of RPL11. Because SRSF1 depletion destabilizes the RPL5-MDM2 interaction, SRSF1 apparently plays a similar role as RPL11. SRSF1 might replace RPL11 in one of the complexes, perhaps in response to particular stress signals. It will be interesting to investigate whether the different complexes are redundant or activate stress responses varying in magnitude or precise outcome. Furthermore, considering that SRSF1 recruits the RP-MDM2 complex to limit its own aberrant activity, this may be a generic mechanism that other oncogenic SR proteins perhaps also adopt to limit the consequences of their own overexpression. The hallmarks of SRSF1-induced senescence are distinct from most OIS pathways described to date (Fig. 1). Classical OIS, as described for other oncogenes, such as H-ras V12, is primarily a DNA-damage response induced by hyper-proliferation and oxidative stress.1 SRSF1-induced senescence, on the other hand, proceeds rapidly in the absence of hyper-proliferation or DNA damage. Furthermore, we did not observe induction of the cell-cycle regulators Rb or ARF/p14, which play critical roles in regulating Ras-induced senescence and MYC-induced apoptosis. Although SRSF1-induced senescence shares common features with PTEN-loss-induced cellular senescence and the related Akt-induced senescence,7 unlike the latter it does not require mTOR for p53 activation. Thus, we have identified a new OIS mechanism that relies on cross-talk between spliceosomal and ribosomal components. Figure 1. SRSF1-induced senescence is mechanistically distinct from classical oncogene-induced senescence. Our results indicate that p53 inactivation is likely a pre-requisite for SRSF1-driven tumorigenesis. About 50% of human tumors bear missense mutations in TP53. In addition, tumors can also harbor lesions in regulators of p53 expression, such as MDM2 amplification or ARF deletion. An intriguing scenario is that SRSF1 itself may acquire mutations, so as to prevent its association with MDM2. SRSF1-overexpressing cells might also escape OIS by accumulating oncogenic mutations in TP53,8 in which case SRSF1-mediated stabilization of mutant p53 would show oncogenic cooperation, leading to a more aggressive phenotype. Thus, though our findings emphasize the potential for regression of SRSF1-dependent tumors by anti-cancer therapies aimed at reactivating the p53-tumor suppressor pathway, they also reinforce the need for molecular characterization of tumors so as to adopt suitable therapies. In summary, our recent publication highlights a novel OIS mechanism that identifies the regulators of the ribosomal-stress response as key players in this tumor-protective pathway. Whether this is unique to SRSF1 activation, or is a conserved function of the RPL-MDM2 complexes remains to be explored. However, it is clear that SRSF1 not only functions as a mediator of ribosomal stress, but also utilizes this mechanism to add another layer to its autoregulation. Furthermore, our study implicates spliceosomal and ribosomal components in non-canonical roles as regulators of a pathway critical for maintenance of cellular homeostasis, further emphasizing the inherent complexity of these key cellular processes.

Serine/Arginine Splicing Factor 1 (SRSF1) is the archetype member of the SR protein family of splicing regulators. Since its discovery over two decades ago, SRSF1 has been repeatedly surprising and intriguing investigators by the plethora of complex biologic pathways it regulates. These include several key aspects of mRNA metabolism, such as mRNA splicing, stability, and translation, as well as other mRNA-independent processes, such as miRNA processing, protein sumoylation, and the nucleolar stress response. In this review, the structural features of SRSF1 are discussed as they relate to the intricate mechanism of splicing and the multiplicity of functions it performs. Similarly, a list of relevant alternatively spliced transcripts and SRSF1 interacting proteins is provided. Finally, emphasis is given to the deleterious consequences of overexpression of the SRSF1 proto-oncogene in human cancers, and the complex mechanisms and pathways underlying SRSF1-mediated transformation. The accumulated knowledge about SRSF1 provides critical insight into the integral role it plays in maintaining cellular homeostasis and suggests new targets for anticancer therapy. Mol Cancer Res; 12(9); 1195–204. ©2014 AACR.

Alternative splicing is a key control point in gene expression, whose misregulation contributes to cancer malignancy. Although certain splicing factors (SFs) and their targets are altered in human tumors, the functional significance of these alterations remains unclear. We previously demonstrated that the splicing factor SRSF1 is upregulated in human breast tumors and promotes transformation in vivo and in vitro. SRSF1 is a prototypical member of the SR protein family, composed of 12 structurally related proteins. However, little is known about differences and redundancies in their splicing targets and biological functions. Here, we investigated whether additional SFs also promoted breast cancer, using transformation models that mimic the relevant biological context. In parallel, we used RNA sequencing (RNA-seq) to systematically identify their oncogenic splicing targets. By mining a large collection of human tumors from the TCGA project, we defined the molecular portraits of SFs alterations in breast tumors. We identified five SFs amplified and/or overexpressed in at least 10% of breast tumors. We then used SF-overexpressing human mammary epithelial MCF-10A cells grown in organotypic 3-D culture; these cells form polarized growth-arrested acinar structures, similar to the terminal units of mammary ducts. Various breast-cancer oncogenes are known to disrupt acinar growth and/or architecture. Interestingly, only certain SFs were oncogenic in this context, differentially affecting cell proliferation, apoptosis, or acinar organization, suggesting non-redundant functions. We then characterized the splicing targets relevant for SF-mediated transformation. We developed a bioinformatics pipeline to identify and quantify splicing variation in RNA-seq data. We defined the global repertoire of SF-regulated splicing events in 3-D culture and compared the target specificities of various SR proteins. In addition, we identified splicing targets regulated both in 3-D culture as well as in human breast tumors. Strikingly, SFs that promoted similar phenotypic changes shared a significant number of splicing targets, suggesting that they regulate common genes to promote tumor initiation. Furthermore, specific SFs affected targets previously associated with epithelial to mesenchymal transition, and increased cell migration or invasion. Finally, we uncovered that the splicing regulator TRA2β is required for the maintenance of metastatic properties of human breast-cancer cells in 3-D culture and in mouse orthotopic models. Furthermore, TRA2β levels correlate with increased metastatic incidence in breast cancer patients. Thus TRA2β represent a potential target for therapeutics development. In summary, we gained new insights into the biological functions of SR proteins and identified novel oncogenic SF-regulated splicing events involved in tumor initiation and metastasis. Citation Format: Olga Anczuków, Shipra Das, Kuan-Ting Lin, Jie Wu, Martin Akerman, Senthil K. Muthuswamy, Adrian R. Krainer. Nonredundant functions of splicing factors in breast-cancer initiation and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A50.

Background: Medical university examinations are known to cause mental stress. Stress can lead to changes in the normal functioning of the human body. Objectives: This study was done among the 1st professional MBBS students of SMIMS to determine the effect of university examination on vitals and differential leucocyte count (DLC). Material and Methods: 100 students, 26 male and 74 female, aged 18 to 21 years, were randomly assessed before and during their 1st professional university examination. Total of 13 Students suffering from fever, hypertension and on long term medication were excluded. 87 students were subjected to clinical check-up (Blood Pressure & Heart Rate) and estimation of DLC by staining the blood smear with Leishman’s Stain. Results: Data were analyzed and compared with pre-examination results. 85% students were having significant increase in neutrophil count, heart rate and systolic blood pressure. However, Eosinophil, Lymphocytes and Monocytes counts were found to be decreased. Conclusion: Examinations in medical school are stressful enough to produce changes in heart rate, blood pressure and differential leucocytes counts although all the students were in good health status. Key Words: Stress, Examination, DLC, Blood Pressure, Heart Rate

The splicing-factor oncoprotein SRSF1 (also known as SF2/ASF or ASF/SF2) is upregulated in breast cancers. We investigated the ability of SRSF1 to transform human and mouse mammary epithelial cells in vivo and in vitro. SRSF1-overexpressing COMMA-1D cells formed tumors, following orthotopic transplantation to reconstitute the mammary gland. In three-dimensional (3D) culture, SRSF1-overexpressing MCF-10A cells formed larger acini than control cells, reflecting increased proliferation and delayed apoptosis during acinar morphogenesis. These effects required the first RNA-recognition motif and nuclear functions of SRSF1. SRSF1 overexpression promoted alternative splicing of BIM (also known as BCL2L11) and BIN1 to produce isoforms that lack pro-apoptotic functions and contribute to the phenotype. Finally, SRSF1 cooperated specifically with MYC to transform mammary epithelial cells, in part by potentiating eIF4E activation, and these cooperating oncogenes are significantly coexpressed in human breast tumors. Thus, SRSF1 can promote breast cancer, and SRSF1 itself or its downstream effectors may be valuable targets for the development of therapeutics.

Cancer cells display aberrant alternative splicing profiles, leading to the production of isoforms that can increase cell proliferation and migration, 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. Changes in the expression of splicing factors have been reported in various types of cancers, including breast. We demonstrated previously that the splicing factor SRSF1 is 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 whether additional splicing factors also promote mammary epithelial cell transformation by using models that mimic the correct biological context in which tumors arise. We first identified splicing factors overexpressed at the transcript and protein levels in a large collection of human breast tumors and cancer cell lines. We then compared the ability of 10 selected splicing factors to transform human mammary epithelial MCF-10A cells grown in organotypic 3-D culture. These 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 breast cancer oncogenes are known to disrupt acinar growth and/or architecture. We assessed how splicing-factor overexpression affects differentially 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, without promoting transformation. We are now characterizing the splicing targets relevant for specific splicing-factor-mediated transformation by next-generation RNA-sequencing. Furthermore, we are investigating which splicing factors are necessary or sufficient to promote metastatic properties of 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 co-expressed with MYC in human breast tumors. In conclusion, by identifying oncogenic splicing factors, their targets and their regulators, we hope to establish novel targets for targeted cancer therapies. Citation Format: Olga Anczukow, Kuan-Ting Lin, Shipra Das, Jie Wu, Martin Akerman, Senthil K. Muthuswamy, Adrian R. Krainer. Differential functions of splicing factors in breast cancer initiation and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A078.

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.