The 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 co-expressed in lung carcinomas, and MYC knockdown downregulates SRSF1 expression in lung-cancer cell lines. MYC directly activates transcription of SRSF1 through two non-canonical 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.
Splicing abnormalities frequently occur in cancer. A key role as splice site choice regulator is played by the members of the SR (Ser/Arg-rich) family of proteins. We recently demonstrated that SRSF2 is involved in cisplatin-mediated apoptosis of human lung carcinoma cell lines. In this study, by using immunohistochemistry, we demonstrate that the SR proteins SRSF1 and SRSF2 are overexpressed in 63% and 65% of lung adenocarcinoma (ADC) as well as in 68% and 91% of squamous cell lung carcinoma (SCC), respectively, compared to normal lung epithelial cells. In addition, we show that SRSF2 overexpression correlates with high level of phosphorylated SRSF2 in both ADC (p<0.0001) and SCC (p = 0.02), indicating that SRSF2 mostly accumulates under a phosphorylated form in lung tumors. Consistently, we further show that the SR-phosphorylating kinases SRPK1 and SRPK2 are upregulated in 92% and 94% of ADC as well as in 72% and 68% of SCC, respectively. P-SRSF2 and SRPK2 scores are correlated in ADC (p = 0.01). Using lung adenocarcinoma cell lines, we demonstrate that SRSF1 overexpression leads to a more invasive phenotype, evidenced by activation of PI3K/AKT and p42/44MAPK signaling pathways, increased growth capacity in soft agar, acquisition of mesenchymal markers such as E cadherin loss, vimentin and fibronectin gain, and increased resistance to chemotherapies. Finally, we provide evidence that high levels of SRSF1 and P-SRSF2 proteins are associated with extensive stage (III–IV) in ADC. Taken together, these results indicate that a global deregulation of pre-mRNA splicing regulators occurs during lung tumorigenesis and does not predict same outcome in both Non Small Cell Lung Carcinoma histological sub-types, likely contributing to a more aggressive phenotype in adenocarcinoma.
Up-regulation of the apoptosis-regulatory gene Mcl-1 (myeloid cell leukemia-1) occurs in different cancer types and is linked with drug resistance to cancer therapies. It is well known that Mcl-1 pre-mRNA undergoes alternative splicing events to produce two functionally distinct proteins, Mcl-1S (pro-apoptotic) and Mcl-lL (anti-apoptotic); the latter isoform is predominant in different cancers including breast and ovarian cancer cells. In the present study we report that the RNA-binding protein (RBP) and proto-oncogene SRSF1 (serine and arginine-rich splicing factor 1) influences splicing of Mcl-1 in both MCF-7 and MDA-MB-231 breast cancer cells and JAR choriocarcinoma cells; we also show for the first time that another RBP SRSF5 affects splicing of Mcl-1 in the MCF-7 cells. Moreover, we report that SRSF1 is involved in other aspects of Mcl-1 regulation with knockdown of SRSF1, by RNAi, resulting in a significant decrease in Mcl-1 protein levels in MCF-7 cells but an increase in JAR cells, respectively, by potentially affecting protein stability and translation of Mcl-l. The key findings from this study highlight the importance of the cellular context of different cancer cells for the function of multifunctional RBPs like SRSF1 and have implications for therapeutic approaches employed to target Mcl-1.
Serine/arginine-rich splicing factor 1 (SRSF1), previously designated SF2/ASF, belongs to a family of SR proteins that regulate constitutive and alternative splicing. SRSF1 expression is increased in tumors from several tissues and elicits changes in key target genes involved in tumor genesis. Several protein kinases phosphorylate SRSF1, which regulates its localization and function. It is previously reported that protein kinase A (PKA) phosphorylates SRSF1, but the importance of this modification is not well characterized. Here, we show that PKA phosphorylates SRSF1 on serine 119 in vitro. Phosphorylation of SRSF1 on this site enhanced the RNA binding capacity of SRSF1 in vivo and reduced the protein’s capacity to activate splicing of the Minx transcript in vitro. We also confirm an interaction between SRSF1 and PKA Cα1 and demonstrate that this interaction is not dependent on serine 119 phosphorylation but requires active PKA Cα1. We conclude that PKA phosphorylation of SRSF1 at serine 119 regulates SFRS1-dependent RNA binding and processing but not its interaction with PKA.
pre-mRNA splicing regulation; SRSF1; PKA; phosphorylation
Acute lymphoblastic leukemia (ALL) is the most frequently-occurring malignant neoplasm in children, but the pathogenesis of the disease remains unclear. In a microarray assay using samples from 100 children with ALL, SFRS1 was found to be up-regulated. Serine/arginine-rich splicing factor 1 (SRSF1, also termed SF2/ASF), encoded by the SFRS1 gene, had been shown to be a pro-oncoprotein. Our previous study indicated that SRSF1 can be methylated by protein arginine methyltransferase 1 (PRMT1) in vitro; however, the biological function of SRSF1 and PRMT1 in pediatric ALL are presently unknown.
Matched, newly diagnosed (ND), complete remission (CR) and relapse (RE) bone marrow samples from 57 patients were collected in order to evaluate the expression patterns of SRSF1 and PRMT1. The potential oncogenic mechanism of SRSF1 and PRMT1 in leukemogenesis was also investigated.
We identified significant up-regulation of SRSF1 and PRMT1 in the ND samples. Importantly, the expression of SRSF1 and PRMT1 returned to normal levels after CR, but rebounded in the RE samples. Our observation that SRSF1 could predict disease relapse was of particular interest, although the expression patterns of SRSF1 and PRMT1 were independent of the cytogenetic subtypes. In pre-B-cell lines, both SRSF1 and PRMT1 expression could be efficiently attenuated by the clinical chemotherapy agents arabinoside cytosine (Ara-c) or vincristine (VCR). Moreover, SRSF1 and PRMT1 were associated with each other in leukemia cells in vivo. Knock-down of SRSF1 resulted in an increase in early apoptosis, which could be further induced by chemotherapeutics.
Our results indicate that SRSF1 serves as an anti-apoptotic factor and potentially contributes to leukemogenesis in pediatric ALL patients by cooperating with PRMT1.
Acute lymphoblastic leukemia; Splicing factor SRSF1; Protein arginine methyltransferase 1 (PRMT1); Alternative splicing; Arginine methylation
Angiogenesis is regulated by the balance of pro-angiogenic VEGF165 and anti-angiogenic VEGF165b splice isoforms. Mutations in WT1, the Wilms’ tumour suppressor gene, suppress VEGF165b and cause abnormal gonadogenesis, renal failure and Wilms’ tumours. In WT1 mutant cells, reduced VEGF165b was due to lack of WT1 mediated transcriptional repression of the splicing factor kinase SRPK1. WT1 bound to the SRPK1 promoter, and repressed expression through a specific WT1 binding-site. In WT1 mutant cells SRPK1-mediated hyperphosphorylation of the oncogenic RNA binding protein SRSF1 regulated splicing of VEGF, and rendered WT1 mutant cells pro-angiogenic. Altered VEGF splicing was reversed by wildtype WT1, knockdown of SRSF1 or SRPK1 and inhibition of SRPK1, which prevented in vitro and in vivo angiogenesis and associated tumour growth.
SR proteins promote spliceosome formation by recognizing exonic splicing enhancers (ESEs) during pre-mRNA splicing. Each SR protein binds diverse ESEs using strategies that are yet to be elucidated. Here, we show that the RNA-binding domain (RBD) of SRSF1 optimally binds to decameric purine rich ESE sequences although locations of purines are not stringently specified. The presence of uracils either within or outside of the recognition site is detrimental for binding with SRSF1. The entire RBD, comprised of two RRMs and a glycine-rich linker, is essential for ESE binding. Mutation within each segment reduced or nearly abolished binding, suggesting that these segments mediate cooperative binding. The linker plays a decisive role in organizing ESE binding. The flanking basic regions of the linker appear to communicate with each other in bringing the two RRMs close together to form the complex with RNA. Our study thus suggests semi-conservative adaptable interaction between ESE and SRSF1, and such binding mode is not only essential for the recognition of plethora of physiological ESE sequences but may also be essential for the interaction with various factors during the spliceosome assembly.
The SR proteins comprise a family of essential, structurally related RNA binding proteins. The complexity of their RNA targets and specificity of RNA recognition in vivo is not well understood. Here we use iCLIP to globally analyze and compare the RNA binding properties of two SR proteins, SRSF3 and SRSF4, in murine cells.
SRSF3 and SRSF4 binding sites mapped to largely non-overlapping target genes, and in vivo consensus binding motifs were distinct. Interactions with intronless and intron-containing mRNAs as well as non-coding RNAs were detected. Surprisingly, both SR proteins bound to the 3' ends of the majority of intronless histone transcripts, implicating SRSF3 and SRSF4 in histone mRNA metabolism. In contrast, SRSF3 but not SRSF4 specifically bound transcripts encoding numerous RNA binding proteins. Remarkably, SRSF3 was shown to modulate alternative splicing of its own as well as three other transcripts encoding SR proteins. These SRSF3-mediated splicing events led to downregulation of heterologous SR proteins via nonsense-mediated decay.
SRSF3 and SRSF4 display unique RNA binding properties underlying diverse cellular regulatory mechanisms, with shared as well as unique coding and non-coding targets. Importantly, CLIP analysis led to the discovery that SRSF3 cross-regulates the expression of other SR protein family members.
Aging is the major risk factor per se for the development of cardiovascular diseases. The senescence of the endothelial cells (ECs) that line the lumen of blood vessels is the cellular basis for these age-dependent vascular pathologies, including atherosclerosis and hypertension. During their lifespan, ECs may reach a stage of senescence by two different pathways; a replicative one derived from their preprogrammed finite number of cell divisions; and one induced by stress stimuli. Also, certain physiological stimuli, such as transforming growth factor-β, are able to modulate cellular senescence. Currently, the cellular aging process is being widely studied to identify novel molecular markers whose changes correlate with senescence. This review focuses on the regulation of alternative splicing mediated by the serine–arginine splicing factor 1 (SRSF1, or ASF/SF2) during endothelial senescence, a process that is associated with a differential subcellular localization of SRSF1, which typically exhibits a scattered distribution throughout the cytoplasm. Based on its senescence-dependent involvement in alternative splicing, we postulate that SRSF1 is a key marker of EC senescence, regulating the expression of alternative isoforms of target genes such as endoglin (ENG), vascular endothelial growth factor A (VEGFA), tissue factor (T3), or lamin A (LMNA) that integrate in a common molecular senescence program.
alternative splicing; endothelial senescence; SRSF1; endoglin; progerin; VEGF; tissue factor
SR proteins are well known to promote exon inclusion in regulated splicing through exonic splicing enhancers. SR proteins have also been reported to cause exon skipping, but little is known about the mechanism. We previously characterized SRSF1 (SF2/ASF)-dependent exon skipping of the CaMKIIδ gene during heart remodeling. By using mouse embryo fibroblasts derived from conditional SR protein knockout mice, we now show that SR protein-induced exon skipping depends on their prevalent actions on a flanking constitutive exon and requires collaboration of more than one SR protein. These findings, coupled with other established rules for SR proteins, provide a theoretical framework to understand the complex effect of SR protein-regulated splicing in mammalian cells. We further demonstrate that heart-specific CaMKIIδ splicing can be reconstituted in fibroblasts by downregulating SR proteins and upregulating a RBFOX protein and that SR protein overexpression impairs regulated CaMKIIδ splicing and neuronal differentiation in P19 cells, illustrating that SR protein-dependent exon skipping may constitute a key strategy for synergism with other splicing regulators in establishing tissue-specific alternative splicing critical for cell differentiation programs.
Increasing evidence suggests that chromatin modifications have important roles in modulating constitutive or alternative splicing. Here we demonstrate that the PWWP domain of the chromatin-associated protein Psip1/Ledgf can specifically recognize tri-methylated H3K36 and that, like this histone modification, the Psip1 short (p52) isoform is enriched at active genes. We show that the p52, but not the long (p75), isoform of Psip1 co-localizes and interacts with Srsf1 and other proteins involved in mRNA processing. The level of H3K36me3 associated Srsf1 is reduced in Psip1 mutant cells and alternative splicing of specific genes is affected. Moreover, we show altered Srsf1 distribution around the alternatively spliced exons of these genes in Psip1 null cells. We propose that Psip1/p52, through its binding to both chromatin and splicing factors, might act to modulate splicing.
The regulated processing of mRNAs by splicing of exons and introns has the potential to increase the information content of the genome. Various splicing factors have been identified whose binding to cis-acting sequences can influence whether an alternative exon is included or excluded (skipped) in the mature mRNA. However, increasing evidence suggests that the chromatin template also has an important role in modulating splicing. Here we identify a chromatin-associated protein Psip1/Ledgf that can bind to a histone modification enriched at active genes and that can also interact with other proteins involved in mRNA splicing. Loss of Psip1 reduces the chromatin association of specific splicing proteins and alters the pattern of alternative splicing. We propose that Psip1, through its binding to both chromatin and splicing factors, might act to modulate splicing.
SRSF1 splicing factor and nuclear-localized MALAT1 RNA influence the assembly of nuclear speckles. Depletion of SRSF1 compromises the association of splicing factors to nuclear speckles and influences the levels of other SR proteins. SRSF1 regulates RNA polymerase II–mediated transcription.
The mammalian cell nucleus is compartmentalized into nonmembranous subnuclear domains that regulate key nuclear functions. Nuclear speckles are subnuclear domains that contain pre-mRNA processing factors and noncoding RNAs. Many of the nuclear speckle constituents work in concert to coordinate multiple steps of gene expression, including transcription, pre-mRNA processing and mRNA transport. The mechanism that regulates the formation and maintenance of nuclear speckles in the interphase nucleus is poorly understood. In the present study, we provide evidence for the involvement of nuclear speckle resident proteins and RNA components in the organization of nuclear speckles. SR-family splicing factors and their binding partner, long noncoding metastasis-associated lung adenocarcinoma transcript 1 RNA, can nucleate the assembly of nuclear speckles in the interphase nucleus. Depletion of SRSF1 in human cells compromises the association of splicing factors to nuclear speckles and influences the levels and activity of other SR proteins. Furthermore, on a stably integrated reporter gene locus, we demonstrate the role of SRSF1 in RNA polymerase II–mediated transcription. Our results suggest that SR proteins mediate the assembly of nuclear speckles and regulate gene expression by influencing both transcriptional and posttranscriptional activities within the cell nucleus.
Epithelial cells organize into cyst-like structures that contain a spherical monolayer of cells that enclose a central lumen. Using a three-dimensional basement membrane culture model in which mammary epithelial cells form hollow, acinus-like structures, we previously demonstrated that lumen formation is achieved, in part, through apoptosis of centrally localized cells. We demonstrate that the proapoptotic protein Bim may selectively trigger apoptosis of the centrally localized acinar cells, leading to temporally controlled lumen formation. Bim is not detectable during early stages of three-dimensional mammary acinar morphogenesis and is then highly upregulated in all cells of acini, coincident with detection of apoptosis in the centrally localized acinar cells. Inhibition of Bim expression by RNA interference transiently blocks luminal apoptosis and delays lumen formation. Oncogenes that induce acinar luminal filling, such as ErbB2 and v-Src, suppress expression of Bim through a pathway dependent on Erk-mitogen-activated protein kinase; however, HPV 16 E7, an oncogene that stimulates cell proliferation but not luminal filling, is unable to reduce Bim expression. Thus, Bim is a critical regulator of luminal apoptosis during mammary acinar morphogenesis in vitro and may be an important target of oncogenes that disrupt glandular epithelial architecture.
Loss of cell polarity proteins such as Scribble induces neoplasia in Drosophila by promoting uncontrolled proliferation. The role polarity proteins play during tumorigenesis in mammals is poorly understood. We demonstrate that knockdown of Scribble in mammary epithelia disrupts cell polarity, blocks three-dimensional morphogenesis, inhibits apoptosis and induces dysplasia in vivo that progress to tumors after long latency. Knockdown of Scribble also cooperates with oncogenes such as Myc to transform epithelial cells in 3D acini and induce tumors in vivo by blocking activation of an apoptosis pathway. Like knockdown, mislocalization of Scribble from cell-cell junction was sufficient to promote cell transformation. Interestingly, spontaneous mammary tumors in mice and humans possess both downregulated and mislocalized Scribble suggesting a selection-pressure for Scribble inactivation. Thus, we demonstrate that Scribble is a novel regulator of breast cancer and that deregulation of polarity pathways promotes dysplastic and neoplastic growth in mammals by disrupting morphogenesis and inhibiting cell death.
Myc; Scribble; 3D; mammary epithelia; mammary tumorigenesis; mouse models; epithelial organization; polarity; apoptosis; cell architecture
SRSF2 is a prototypical SR protein which plays important roles in the alternative splicing of pre-mRNA. It has been shown to be involved in regulatory pathways for maintaining genomic stability and play important roles in regulating key receptors in the heart. We report here the solution structure of the RNA recognition motifs (RRM) domain of free human SRSF2 (residues 9–101). Compared with other members of the SR protein family, SRSF2 structure has a longer L3 loop region. The conserved aromatic residue in the RNP2 motif is absent in SRSF2. Calorimetric titration shows that the RNA sequence 5′AGCAGAGUA3′ binds SRSF2 with a Kd of 61 ± 1 nM and a 1:1 stoichiometry. NMR and mutagenesis experiments reveal that for SFSF2, the canonical β1 and β3 interactions are themselves not sufficient for effective RNA binding; the additional loop L3 is crucial for RNA complex formation. A comparison is made between the structures of SRSF2–RNA complex with other known RNA complexes of SR proteins. We conclude that interactions involving the L3 loop, N- and C-termini of the RRM domain are collectively important for determining selectivity between the protein and RNA.
Alternative splicing of the pyruvate kinase M gene (PK-M) can generate the M2 isoform and promote aerobic glycolysis and tumor growth. However, the cancer-specific alternative splicing regulation of PK-M is not completely understood. Here, we demonstrate that PK-M is regulated by reciprocal effects on the mutually exclusive exons 9 and 10, such that exon 9 is repressed and exon 10 is activated in cancer cells. Strikingly, exonic, rather than intronic, cis-elements are key determinants of PK-M splicing isoform ratios. Using a systematic sub-exonic duplication approach, we identify a potent exonic splicing enhancer in exon 10, which differs from its homologous counterpart in exon 9 by only two nucleotides. We identify SRSF3 as one of the cognate factors, and show that this serine/arginine-rich protein activates exon 10 and mediates changes in glucose metabolism. These findings provide mechanistic insights into the complex regulation of alternative splicing of a key regulator of the Warburg effect, and also have implications for other genes with a similar pattern of alternative splicing.
alternative splicing; cancer metabolism; pyruvate kinase; SRSF3
Cyclic AMP–dependent protein kinase A (PKA) is required for MCF10A mammary epithelial acinus formation in vitro. PKA plays a dual role by facilitating polarization of cells attached to the extracellular matrix and apoptosis of detached cells.
Epithelial cells form tubular and acinar structures notable for a hollow lumen. In three-dimensional culture utilizing MCF10A mammary epithelial cells, acini form due to integrin-dependent polarization and survival of cells contacting extracellular matrix (ECM), and the apoptosis of inner cells of acini lacking contact with the ECM. In this paper, we report that cyclic AMP (cAMP)-dependent protein kinase A (PKA) promotes acinus formation via two mechanisms. First, cAMP accelerates redistribution of α6-integrin to the periphery of the acinus and thus facilitates the polarization of outer acinar cells. Blocking of α6-integrin function by inhibitory antibody prevents cAMP-dependent polarization. Second, cAMP promotes the death of inner cells occupying the lumen. In the absence of cAMP, apoptosis is delayed, resulting in perturbed luminal clearance. cAMP-dependent apoptosis is accompanied by a posttranscriptional PKA-dependent increase in the proapoptotic protein Bcl-2 interacting mediator of cell death. These data demonstrate that cAMP regulates lumen formation in mammary epithelial cells in vitro, both through acceleration of polarization of outer cells and apoptosis of inner cells of the acinus.
DNA ligase I-deficient 46BR.1G1 cells show a delay in the maturation of replicative intermediates resulting in the accumulation of single- and double-stranded DNA breaks. As a consequence the ataxia telangiectasia mutated protein kinase (ATM) is constitutively phosphorylated at a basal level. Here, we use 46BR.1G1 cells as a model system to study the cell response to chronic replication-dependent DNA damage. Starting from a proteomic approach, we demonstrate that the phosphorylation level of factors controlling constitutive and alternative splicing is affected by the damage elicited by DNA ligase I deficiency. In particular, we show that SRSF1 is hyperphosphorylated in 46BR.1G1 cells compared to control fibroblasts. This hyperphosphorylation can be partially prevented by inhibiting ATM activity with caffeine. Notably, hyperphosphorylation of SRSF1 affects the subnuclear distribution of the protein and the alternative splicing pattern of target genes. We also unveil a modulation of SRSF1 phosphorylation after exposure of MRC-5V1 control fibroblasts to different exogenous sources of DNA damage. Altogether, our observations indicate that a relevant aspect of the cell response to DNA damage involves the post-translational regulation of splicing factor SRSF1 which is associated with a shift in the alternative splicing program of target genes to control cell survival or cell death.
The transcription factor Runx2 is highly expressed in breast cancer cells compared to mammary epithelial cells and contributes to metastasis. Here we directly show that Runx2 expression promotes a tumor cell phenotype of mammary acini in three dimensional culture. Human mammary epithelial cells (MCF-10A) form polarized, growth-arrested acini-like structures with glandular architecture. The ectopic expression of Runx2 disrupts acini formation, and electron microscopic ultrastructural analysis revealed the absence of lumens. Characterization of the disrupted acini structures showed increased cell proliferation (Ki-67 positive cells), decreased apoptosis (Bcl-2 induction) and loss of basement membrane formation (absence of β4-integrin expression). In complementary experiments, inhibition of Runx2 function in metastatic MDA-MB-231 breast cancer cells by stable expression of either shRNA-Runx2 or a mutant Runx2 deficient in subnuclear targeting resulted in reversion of acini to more normal structures and reduced tumor growth in vivo. These novel findings provide direct mechanistic evidence for the biological activity of Runx2, dependent on its subnuclear localization, in promoting early events of breast cancer progression and suggest a molecular therapeutic target.
Runx2; acini; mammary epithelial cells; breast cancer; 3D culture
Gene regulation in response to environmental stress is critical for the survival of all organisms. From Saccharomyces cerevisiae to humans, it has been observed that splicing of mRNA precursors is repressed upon heat shock. However, a mild heat pretreatment often prevents splicing inhibition in response to a subsequent and more severe heat shock, a phenomenon called splicing thermotolerance. We have shown previously that the splicing regulator SRSF10 (formerly SRp38) is specifically dephosphorylated by the phosphatase PP1 in response to heat shock and that dephosphorylated SRSF10 is responsible for splicing repression caused by heat shock. Here we report that a mild heat shock protects SRSF10 from dephosphorylation during a second and more severe heat shock. Furthermore, this “thermotolerance” of SRSF10 phosphorylation, like that of splicing, requires de novo protein synthesis, specifically the synthesis of heat shock proteins. Indeed, overexpression of one of these proteins, Hsp27, inhibits SRSF10 dephosphorylation in response to heat shock and does so by interaction with SRSF10. Our data thus provide evidence that splicing thermotolerance is acquired through maintenance of SRSF10 phosphorylation and that this is mediated at least in part by Hsp27.
Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology with the alternative pre-mRNA processing of caspase 9 as one example. In this study, we delve into the underlying molecular mechanisms that regulate the alternative splicing of caspase 9. Specifically, the pre-mRNA sequence of caspase 9 was analyzed for RNA cis-elements known to interact with SRSF1, a required enhancer for caspase 9 RNA splicing. This analysis revealed thirteen possible RNA cis-elements for interaction with SRSF1 with mutagenesis of these RNA cis-elements identifying a strong intronic splicing enhancer located in intron 6 (C9-I6/ISE). SRSF1 specifically interacted with this sequence, which was required for SRSF1 to act as a splicing enhancer of the inclusion of the four exon cassette. To further determine the biological importance of this mechanism, we employed RNA oligonucleotides to redirect caspase 9 pre-mRNA splicing in favor of caspase 9b expression, which resulted in an increase in the IC50 of non-small cell lung cancer (NSCLC) cells to daunorubicin, cisplatinum, and paclitaxel. In contrast, downregulation of caspase 9b induced a decrease in the the IC50 of these chemotherapeutic drugs. Lastly, these studies demonstrated that caspase 9 RNA splicing was a major mechanism for the synergistic effects of combination therapy with daunorubicin and erlotinib. Overall, we have identified a novel intronic splicing enhancer that regulates caspase 9 RNA splicing and specifically interacts with SRSF1. Furthermore, we demonstrate that the alternative splicing of caspase 9 is an important molecular mechanism with therapeutic relevance to NSCLCs.
ceramide; non-small cell lung cancer; RNA trans-factor; tumor repressor; oncogene; ASF/SF2; SRp30a; SRSF1; chemotherapy; erlotinib; daunorubicin; cisplatinum; paclitaxel
Endoplasmic reticulum (ER) stress signaling can be mediated by the ER kinase PERK, which phosphorylates its substrate eIF2α. This in turn, results in translational repression and the activation of downstream programs that can limit cell growth through cell cycle arrest and/or apoptosis. These responses can also be initiated by perturbations in cell adhesion. Thus, we hypothesized that adhesion-dependent regulation of PERK signaling might determine cell fate. We tested this hypothesis in a model of mammary acini development, a morphogenetic process regulated in part by adhesion signaling. Here we report a novel role for PERK in limiting MCF10A mammary epithelial cell proliferation during acinar morphogenesis in 3D Matrigel culture as well as in preventing mammary tumor formation in vivo. We show that loss of adhesion to a suitable substratum induces PERK-dependent phosphorylation of eIF2α and selective upregulation of ATF4 and GADD153. Further, inhibition of endogenous PERK signaling during acinar morphogenesis, using two dominant-negative PERK mutants (PERK-ΔC or PERK-K618A), does not affect apoptosis but results instead in hyper-proliferative and enlarged lumen-filled acini, devoid of proper architecture. This phenotype correlated with an adhesion-dependent increase in translation initiation, Ki67 staining and upregulation of Laminin-5, ErbB1 and ErbB2 expression. More importantly, the MCF10A cells expressing PERKΔC, but not a vector control, were tumorigenic in vivo upon orthotopic implantation in denuded mouse mammary fat pads. Our results reveal that the PERK pathway is responsive to adhesion-regulated signals and that it is essential for proper acinar morphogenesis and in preventing mammary tumor formation. The possibility that deficiencies in PERK signaling could lead to hyperproliferation of the mammary epithelium and increase the likelihood of tumor formation, is of significance to the understanding of breast cancer.
Hypoxia can regulate many cellular processes. We show that hypoxia, via hypoxia-inducible factor (HIF)-1, blocks anoikis of epithelial cells by activating signaling pathways that inhibit expression of proapoptotic proteins Bim and Bmf. Hypoxia also disrupts mammary morphogenesis and blocks anoikis associated with lumen formation in three-dimensional in vitro model of mammary acini.
Proper adhesion to extracellular matrix is critical for epithelial cell survival. Detachment from matrix signals results in apoptosis, referred to as anoikis. Selective apoptosis of cells that become detached from matrix is associated with the formation of a lumen in three-dimensional mammary epithelial acinar structures in vitro. Because early breast cancer lesions such as carcinoma in situ, characterized by ducts exhibiting lumens filled with cells, are often associated with hypoxic markers, we sought to examine the role of hypoxia in anoikis and lumen formation in mammary epithelial cells. Here, we show that hypoxic conditions inhibit anoikis and block expression of proapoptotic BH3-only family members Bim and Bmf in epithelial cells. Hypoxia-mediated anoikis protection is associated with increased activation of the epidermal growth factor receptor–mitogen-activated protein kinase kinase–extracellular signal-regulated kinase (Erk) kinase pathway and requires the hypoxia-activated transcription factor. Consistent with these data, hypoxic conditions inhibit luminal clearing during morphogenesis in human mammary epithelial acini when grown in three-dimensional cultures and are associated with decreased expression of Bim and Bmf as well as Erk activation. We show that hypoxia regulates specific cell survival pathways that disrupt tissue architecture related to clearing of luminal space during mammary morphogenesis and suggest that hypoxia-mediated anoikis resistance may contribute to cancer progression.
The Rho GTPase Cdc42 is overexpressed and hyperactivated in breast cancer, and several studies have described mechanisms by which it may promote tumor formation and progression. However, little is known about the role of Cdc42 during normal mammary epithelial cell (MEC) morphogenesis. Here we aimed to define the precise role for Cdc42 during primary mammary acinus formation in vitro. For these studies, MECs were isolated from Cdc42fl/fl conditional knockout mice, transduced with Adeno-cre-GFP virus to delete Cdc42 or Adeno-GFP control virus, and effects on morphogenesis were investigated using a three-dimensional (3D) culture assay. Interestingly, markedly fewer mammary acini developed in Cdc42 deficient cultures, and the acini that formed were significantly smaller and disorganized. Cellular proliferation and survival were reduced in the Cdc42 deficient acini. However, control and knockout MECs cultured as monolayers displayed similar cell cycle profiles, suggesting that Cdc42 is important for MEC proliferation in the context of 3D polarity. Overexpression of cyclin D1, which promotes cell cycle progression downstream of Cdc42, failed to rescue the defect in acinus size. Furthermore, lumen formation and apical-basal polarity were disrupted, and mitotic spindle orientation and Cdc42/aPKC polarity complex defects likely contributed to these phenotypes. Studies using dominant negative Cdc42 and siRNa to knockdown Cdc42 in MDcK and Caco-2 cell lines undergoing cystogenesis in 3D cultures revealed critical roles for Cdc42 in spindle orientation, polarity and lumen formation. Our studies, using complete knockout in primary epithelial cells, demonstrate that Cdc42 is not only an important regulator of polarity and lumen formation; it is also essential for proliferation and survival, which are key cellular processes that drive MEC morphogenesis in vitro and in vivo.
Rho GTPase; Cdc42; mammary; morphogenesis; cell polarity; proliferation; apoptosis; three-dimensional culture; epithelial cell; conditional knockout
A high expression of short and immature O-glycans is one of the prominent features of breast cancer cells, which would be attributed to the upregulated expression of glycosyltransferases. Therefore, a detailed elucidation of glycosyltransferases and their substrate(s) may improve our understandings for their roles in mammary carcinogenesis. Here we report that overexpression of polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6), a glycosyltransferase involved in the initial step of O-glycosylation, has transformational potentials through disruptive acinar morphogenesis and cellular changes similar to epithelial-to-mesenchymal transition in normal mammary epithelial cell, MCF10A. As one of the critical O-glycan substrates, we identified fibronectin that was O-glycosylated in vivo and thereby stabilized by GALNT6. Because knockdown of fibronectin abrogated the disruptive proliferation caused by introduction of GALNT6 into epithelial cells, our findings suggest that GALNT6-fibronectin pathway should be a critical component for breast cancer development and progression.