P73 Splicing Research Articles

SCARB1-encoded circ _0029343 induces p73 splicing to promote growth and metastasis of hepatocellular carcinoma via miR-486-5p/SRSF3 axis.

Circular RNAs (circRNAs) exhibit essential regulation in the malignant development of hepatocellular carcinoma (HCC). This study aims to investigate the physiological mechanisms of circ_0029343 encoded by scavenger receptor class B member 1 (SCARB1) involved in the growth and metastasis of HCC. Differentially expressed mRNAs in HCC were obtained, followed by the prediction of target genes of differentially expressed miRNAs and gene ontology and kyoto encyclopedia of genes and genomes analysis on the differentially expressed mRNAs. Moreover, the regulatory relationship between circRNAs encoded by SCARB1 and differentially expressed miRNAs was predicted. In vitro cell experiments were performed to verify the effects of circ_0029343, miR-486-5p, and SRSF3 on the malignant features of HCC cells using the gain- or loss-of-function experiments. Finally, the effects of circ_0029343 on the growth and metastasis of HCC cells in xenograft mouse models were also explored. It was found that miR-486-5p might interact with seven circRNAs encoded by SCARB1, and its possible downstream target gene was SRSF3. Moreover, SRSF3 was associated with the splicing of various RNA. circ_0029343 could sponge miR-486-5p to up-regulate SRSF3 and activate PDGF-PDGFRB (platelet-derived growth factor and its receptor, receptor beta)signaling pathway by inducing p73 splicing, thus promoting the proliferation, migration, and invasion and inhibiting apoptosis of HCC cells. In vivo, animal experiments further confirmed that overexpression of circ_0029343 could promote the growth and metastasis of HCC cells in nude mice. circ_0029343 encoded by SCARB1 may induce p73 splicing and activate thePDGF-PDGFRB signaling pathway through the miR-486-5p/SRSF3 axis, thus promoting the growth and metastasis of HCC cells.

Overexpression of splicing factor poly(rC)-binding protein 1 elicits cycle arrest, apoptosis induction, and p73 splicing in human cervical carcinoma cells.

Splicing factor poly(rC)-binding protein 1 (PCBP1) is a novel tumor suppressor that is downregulated in several cancers thereby regulating tumor formation and metastasis. However, the involvement of PCBP1 in apoptosis of cancer cells and the molecular mechanism remains elusive. On this basis, we sought to investigate the role of splicing factor PCBP1 in the apoptosis in human cervical cancer cells. To investigate PCBP1 functions in vitro, we overexpressed PCBP1 in human cervical cancer cells. A series of cytological function assays were employed to study to the role of PCBP1 in cell proliferation, cell cycle arrest and apoptosis. Overexpression of PCBP1 was found to greatly repress proliferation of HeLa cells in a time-dependent manner. It also induced a significant increase in G2/M phase arrest and apoptosis. Furthermore, overexpressed PCBP1 favored the production of long isoforms of p73, thereby inducing upregulated ratio of Bax/Bcl-2, the release of cytochrome c and the expression of caspase-3. Our results revealed that PCBP1 played a vital role in p73 splicing, cycle arrest and apoptosis induction in human cervical carcinoma cells. Targeting PCBP1 may be a potential therapeutic strategy for cervical cancer therapy.

Inhibition of SF3b1 by pladienolide B evokes cycle arrest, apoptosis induction and p73 splicing in human cervical carcinoma cells

Pladienolide B is a potent cancer cell growth inhibitor that targets the SF3b1 subunit of the spliceosome. There is considerable interest in the compound as a tool to study SF3b1 function in cancer. However, so far little information is available on the molecular mechanism of SF3b1 eliciting apoptosis in cancer cells. Here, we investigated the molecular mechanism of SF3b1 eliciting apoptosis in human cervical carcinoma cells. We demonstrated that inhibition of SF3b1 by pladienolide B inhibited proliferation of HeLa cells at low nanomolar concentrations in a dose- and time-dependent manner. It also induced G2/M phase arrest and significant rise of apoptotic cells. Moreover, it is indicated that inhibition of SF3b1 by pladienolide B induced Tap73/ΔNp73 expression and consequently down-regulated Bax/Bcl-2 ratio, cytochrome c release and caspase-3 expression. Thus, our results showed that SF3b1 plays a pivotal role in cycle arrest, apoptosis induction, and p73 splicing in human cervical carcinoma cells, suggesting that SF3b1 could be used as a potential candidate for cervical cancer therapy.

TAp73β-mediated suppression of cell migration requires p57Kip2 control of actin cytoskeleton dynamics

The TP73 gene, a member of the p53 family, due to the use of different promoters and alternative splicing, is transcribed into different isoforms with contrasting attributes and which contribute to its functional diversity. Considerable efforts are made to identify the functional diversity of the p73 splicing variants during tumorigenesis. TAp73α and TAp73β isoforms have been shown to differentially regulate cell cycle progression, differentiation and apoptosis. Interestingly, a particular increase in expression of the TAp73 isoform, in favor of the α splicing variant, has been reported in multiple tumour types. Here, we report a distinctive role for TAp73β isoform in the control of cell migration and invasion. In fact, TAp73β- dependent induction of p57(Kip2) expression accounted for inhibitory effects on the actin cytoskeleton dynamics and thereby cancer cell motility. In contrast, TAp73α is not able to induce p57(Kip2) expression, and exhibits a positive effect on actin cytoskeleton dynamics as well as cell migration and invasion. In conclusion, the inhibitory effect on cell migration and invasion of TAp73β would qualify this distinct p73 isoform as tumor suppressor gene. In contrast, the promoting effect of TAp73α on cell motility and invasion strengthens the potential oncogenic activities of this p73 isoform.

Antisense gapmers selectively suppress individual oncogenic p73 splice isoforms and inhibit tumor growth in vivo

BackgroundDifferential mRNA splicing and alternative promoter usage of the TP73 gene results in the expression of multiple NH2-truncated isoforms that act as oncogenes. Abundant levels of these p73 variants in a variety of human cancers correlated with adverse clinical prognosis and response failure to conventional therapies, underscoring their relevance as marker for disease severity and target for cancer intervention. With respect to an equally important role for amino-truncated p73 splice forms (ΔTAp73) and ΔNp73 (summarized as DNp73) in the tumorigenic process, we designed locked nucleic acid (LNA) antisense oligonucleotide (ASO) gapmers against individual species that were complementary to ΔEx2 and ΔEx2/3 splice junctions and a region in exon 3B unique for ΔN' and ΔN.ResultsTreatment of cancer cells with these ASOs resulted in a strong and specific reduction of tumorigenic p73 transcripts and proteins, importantly, without abolishing the wild-type p73 tumor suppressor form as observed with p73-shRNA. The specific antisense oligonucleotides rescued cells from apoptosis inhibition due to overexpression of their corresponding amino-truncated p73 isoform and decreased tumor cell proliferation. Furthermore, ASO-116 against ΔEx2/3 coupled to magnetic nanobead polyethyleneimine (MNB/PEI) carriers significantly inhibited malignant melanoma growth, which correlated with a shift in the balance between endogenous TAp73 and ΔEx2/3 towards apoptotic full-length p73.ConclusionOur study demonstrates the successful development of LNA-ASOs that selectively differentiate between the closely related p73 oncoproteins, and provide new tools to further delineate their biological properties in different human malignancies and for therapeutic cancer targeting.

TIP60 Represses Transcriptional Activity of p73β via an MDM2-bridged Ternary Complex

TIP60, a histone acetyl transferase, acts as a p53 coactivator by interfering with MDM2-mediated degradation of p53. However, little is known about its functional regulation of p73, which has structural features similar to p53. In this study we found that TIP60 represses apoptosis, which is induced by exogenous and endogenous p73beta. TIP60 also negatively regulated the expression of p73beta downstream target genes such as p21 and Bax. Moreover, the specific repression of p73beta-mediated transactivation by TIP60 was independent of p53 expression and not due to histone deacetylase recruiting transcriptional machinery. Transcriptional activities of both p73 splicing variants, p73alpha and p73beta, were also repressed by TIP60. Furthermore, TIP60 markedly enhanced p73beta binding affinity to MDM2 and physically associated with MDM2 through its zinc finger domain, which is specifically localized in the nucleus. Therefore, we demonstrate that TIP60 forms a ternary complex with p73beta, which is directly bridged by MDM2. It is important to note that our findings contribute to a functional linkage between TIP60 and p73beta through MDM2 in the transcriptional regulation of cellular apoptosis.

TAp73 isoforms antagonize Notch signalling in SH‐SY5Y neuroblastomas and in primary neurones

p73, like Notch, has been implicated in neurodevelopment and in the maintenance of the mature central nervous system. In this study, by the use of reporter-gene assays, we demonstrate that C-promoter binding factor-1 (CBF-1)-dependent gene transcription driven by the Notch-1 intracellular domain (N1(ICD)) is potently antagonized by exogenously expressed transactivating (TA) p73 splice variants in SH-SY5Y neuroblastomas and in primary neurones. Time course analysis indicated that the inhibitory effects of TAp73 are direct and are not mediated via the product of a downstream target gene. We found that endogenous TAp73 stabilized by either c-Abl or cisplatin treatment also potently antagonized N1(ICD)/CBF-1-dependent gene transcription. Furthermore, western blotting revealed that exogenous TAp73 suppressed endogenous hairy and enhancer of split-1 (HES-1) protein levels and antagonized the increase in HES-1 protein induced by exogenous N1(ICD) expression. Evidence of a direct physical interaction between N1(ICD) and TAp73alpha was demonstrated by co-immunoprecipitation. Using Notch deletion constructs, we demonstrate that TAp73alpha binds the N1(ICD) in a region C-terminal of aa 2094. Interestingly, DeltaNp73alpha and TAp73alpha(R292H) also co-purified with N1(ICD), but neither inhibited N1(ICD)/CBF-1-dependent transcription. This suggests that an intact transactivation (TA) domain and the ability to bind DNA are necessary for TAp73 to antagonize Notch signalling. Finally we found that TAp73alpha reversed the N1(ICD)-mediated repression of retinoic acid-induced differentiation of SH-SY5Y neuroblastomas, providing functional evidence for an inhibitory effect of TAp73alpha on notch signalling. Collectively, these findings may have ramifications for neurodevelopment, neurodegeneration and oncogenesis.

Full-length p73α Represses Drug-induced Apoptosis in Small Cell Lung Carcinoma Cells

The p73 gene, a member of the p53 family, encodes several variants through differential splicing and use of alternative promoters. At the NH2 terminus, two different promoters generate the full-length and the DeltaN isoforms, with or without the transactivating domain. At the COOH terminus, seven isoforms generated through alternative splicing have been cloned. Previous studies have demonstrated that DeltaNp73 isoforms exert a dominant-negative effect on p73 by blocking their transactivation activity and hence the ability to induce apoptosis. Considerable efforts are made to identify the functional diversity of the COOH-terminal p73 variants. In this study, we found that p73alpha inhibited drug-induced apoptosis in small cell lung carcinoma cells, whereas p73beta promoted it. p73alpha prevented Bax activation, mitochondrial dysfunction, and caspase activation. In addition, p73alpha was also able to reduce apoptosis induced by the BH3-only protein PUMA (p53 up-regulated modulator of apoptosis). Furthermore, we discovered that p73alpha is able to inhibit the pro-apoptotic effect of p73beta, demonstrating the existence of equilibrium between these two p73 isoforms. In conclusion, the reported overexpression of p73alpha in certain tumor types, and our findings that p73alpha exerts anti-apoptotic functions, indicate a potential oncogenic activity for p73.

RT-PCR analysis of p73 splice variants, ease or tease?

RT-PCR analysis of p73 splice variants, ease or tease?

Ectopic Expression of p73α, but Not p73β, Suppresses Myogenic Differentiation

The TRP73 gene, a member of the p53 family, encodes several variants through differential splicing and use of alternative promoters. At the N terminus, two different promoters generate the full-length and the DeltaN isoforms, with or without the transactivating domain. At the C terminus, seven isoforms generated through alternative splicing have been cloned. Previous studies have demonstrated that DeltaN-p73 interferes with p73-induced apoptosis. However, there has been no evidence for functional diversity of the C-terminal p73 variants. In this study, we found that p73alpha and p73beta exerted differential effect on the differentiation of C2C12 myoblasts. Although p73beta lacked any detectable effect on differentiation, p73alpha caused a substantial delay in the expression of muscle-specific genes. In co-transfection experiments p73alpha, but not p73beta, attenuated the transcriptional activity of MyoD. Microarray-based gene profiling confirmed the protraction of MyoD-dependent gene expression in C2C12 cells stably expressing p73alpha. Notwithstanding the differential effect on differentiation, p73alpha and p73beta showed similar activity in sensitizing C2C12 myoblasts to cisplatin-induced cell death. These results demonstrated a functional diversity between the two C-terminal variants of p73 and suggested that p73alpha can regulate cellular differentiation in addition to its role in stimulating cell death.

Expression of P73 Splice Variants Correlates to Resistance to DNA Damaging Drugs in Childhood T-Lineage Acute Lymphoblastic Leukemia.

Children with T-lineage Acute Lymphoblastic Leukemia (T-ALL) have a higher relapse-risk and are in-vitro more resistant to therapeutic drugs compared to ALL patients with a precursor-B phenotype. Cellular resistance to anti-cancer agents has previously shown to be associated with failure of P53 family member signaling by abrogation of P53 function due to loss-of-function mutations or dominant-negative inhibition by isoforms of P73 lacking (part of) the N-terminal transactivation domain (P73ΔEX2, P73ΔEX2/3, ΔN-P73 and ΔN'-P73). Since p53 mutations are not commonly found in T-ALL, we investigated the expression levels of p73 splice variants in relation to drug resistance in children with T-ALL. Splice variants were quantitatively measured at the mRNA level in leukemic cells of 55 T-ALL patients and mononuclear cells of 12 non-leukemic controls. TA-p73 (transactivation competent), p73Δex2, p73Δex2/3, ΔN-p73 and ΔN'-p73 were all found to be present at a relatively higher mRNA level in T-ALL patients than controls (P < 0.05 for all), suggesting that expression of the TP73 gene is deregulated in T-ALL. Resistance of T-ALL cells to the DNA damaging drug daunorubicin correlated with mRNA levels of the dominant-negative variants of p73, i.e. ΔN-p73 and ΔN'-p73 (Rs = 0.38, P = 0.03). In contrast, expression of none of the variants, including ΔN-p73 and ΔN'-p73, was related to resistance of T-ALL cells to non-DNA damaging drugs (prednisolone, vincristine and L-asparaginase). In conclusion, high expression of ΔN-p73 and ΔN'-p73 variants possibly contributes to resistance to DNA damaging drugs in childhood T-ALL.

Transcriptional activities of p73 splicing variants are regulated by inter-variant association

p73 has been identified as a gene that encodes a protein with significant identity with the tumour suppressor p53. The main structural difference between p73 and p53 is the additional C-terminal region of p73. Six isoforms of p73 with differing C-terminal structures, α, β, γ, δ, ∊ and ξ, have been reported. These variants differ in transcriptional activity on p53-responsive promoters. Here we report a possible mechanism of transcriptional activation by p73 splicing variants. C-terminal deletion mutants of p73α showed a significantly higher level of transcriptional activity than wild-type p73α, suggesting that the C-terminal structure of p73α functions to repress the transcriptional activity of p73α. The results of immunoprecipitation assays and two-hybrid assays in mammalian cells showed that the p73 variants interacted with each other, but not with p53. The transcriptional activity of p73β was reduced by co-expression with either p73α or p73∊, which bears an identical C-terminal structure to p73α. Co-expression of the C-terminal portion of p73α or p73∊ with p73β also resulted in reduced transcriptional activity. Moreover, we observed that the level of endogenous p21 protein induced by p73β was decreased by co-expression of full-length p73∊ or the C-terminal region of p73α or p73∊. These observations suggest that p73-mediated gene expression is regulated by the interactions of p73 splicing variants in the cell.

Transcriptional activities of p73 splicing variants are regulated by inter-variant association.

p73 has been identified as a gene that encodes a protein with significant identity with the tumour suppressor p53. The main structural difference between p73 and p53 is the additional C-terminal region of p73. Six isoforms of p73 with differing C-terminal structures, alpha, beta, gamma, delta, epsilon and xi, have been reported. These variants differ in transcriptional activity on p53-responsive promoters. Here we report a possible mechanism of transcriptional activation by p73 splicing variants. C-terminal deletion mutants of p73 alpha showed a significantly higher level of transcriptional activity than wild-type p73 alpha, suggesting that the C-terminal structure of p73 alpha functions to repress the transcriptional activity of p73 alpha. The results of immunoprecipitation assays and two-hybrid assays in mammalian cells showed that the p73 variants interacted with each other, but not with p53. The transcriptional activity of p73 beta was reduced by co-expression with either p73 alpha or p73 epsilon, which bears an identical C-terminal structure to p73 alpha. Co-expression of the C-terminal portion of p73 alpha or p73 epsilon with p73 beta also resulted in reduced transcriptional activity. Moreover, we observed that the level of endogenous p21 protein induced by p73 beta was decreased by co-expression of full-length p73 epsilon or the C-terminal region of p73 alpha or p73 epsilon. These observations suggest that p73-mediated gene expression is regulated by the interactions of p73 splicing variants in the cell.

Differential expression of p73 splice variants and protein in benign and malignant ovarian tumours

Differential expression of p73 splice variants and protein in benign and malignant ovarian tumours

Differential expression of p73 splice variants and protein in benign and malignant ovarian tumours

The p73 gene encodes a protein with substantial structural and functional similarities to the tumour-suppressor p53. Alternative splicing of p73 mRNA leads to expression of 6 known RNA species and proteins (alpha, beta, gamma, delta, epsilon, zeta). We analysed the expression of these splice variants in ovarian adenocarcinoma by RT-PCR followed by detection of amplicons with the Southern technique and by immunoblot in 32 malignant and benign epithelial ovarian tumour specimens and 3 ovarian adenocarcinoma cell lines (A2780, 2008, OVCAR-3). p73alpha mRNA was expressed in all 17 ovarian cancer specimens, and 14 of 17 expressed at least 3 splice variants. In contrast, a different expression pattern was present in the ovarian adenomas: p73alpha was detected in 6 of 12 benign tumours, and only 1 adenoma expressed 3 splice variants. p73 protein was expressed in 9 of 16 ovarian cancer specimens, in all cell lines and in 1 of 3 borderline tumours. In contrast, none of 9 ovarian adenomas expressed detectable amounts of p73 protein. Expression of p73 mRNA and protein was not correlated with FIGO stage and histological grade, but we observed a significant correlation with over-expression of p53 protein. In summary, epithelial ovarian cancers express a more complex p73 isoform pattern and higher levels of p73 mRNA and protein than ovarian adenomas.

Two new p73 splice variants, gamma and delta, with different transcriptional activity.

p73 has been recently identified as a new structural and functional homologue of the transcription factor p53. It is expressed in either a full-length form, alpha, or a shorter beta mRNA variant, with exon 13 spliced out. Here we report the identification and functional characterization of two new p73 splicing variants, gamma (splicing out exon 11) and delta (splicing out exons 11, 12, and 13). Both gamma and delta p73 variants are expressed in human peripheral blood lymphocytes, primary keratinocytes, and different tumor cell lines, including neuroblastoma, glioblastoma, melanoma, hepatoma, and leukemia. The expression pattern of the four p73 splicing variants differs in both primary cells of different lineage and established cell lines even within the same type of tumor. A two-hybrid assay was used to characterize the homodimeric and heterodimeric interactions between the p73 variants, and showed that neither p73gamma nor p73delta interact with p53, whereas p73gamma showed strong interactions with all p73 isoforms, and p73delta binds efficiently p73alpha and p73gamma but only weakly p73beta. At the functional level, p73gamma is significantly less efficient in activating transcription of the p21(Waf1/Cip1) promoter than p53 or p73beta, whereas the effect of p73delta is intermediate and comparable to that of p73alpha. The ability of the different p73 variants to affect cell growth in p53 null osteosarcoma SAOS-2 cells correlates with their transcriptional activity on the p21(Waf1/Cip1) promoter: p73beta is the most efficient in inhibiting colony formation, whereas p73gamma is almost ineffective. Our results suggest that p73 isoforms may be differentially regulated, with four different isoforms capable of interacting among themselves and with p53. The relative expression level of each splice variant may modulate p73 transcriptional and growth suppression activities by affecting heterodimer formation.