Isoform-specific Disruption of the TP73 Gene Reveals a Critical Role for TAp73gamma in Tumorigenesis via Leptin

TP73, a member of the p53 family, is expressed as TAp73 and DNp73 along with multiple C-terminal isoforms (a-n). DNp73 is primarily expressed in neuronal cells and necessary for neuronal development. Interestingly, while TAp73a is a tumor suppressor and predominantly expressed in normal cells, TAp73 is found to be frequently altered in human cancers, suggesting a role of TAp73 C-terminal isoforms in tumorigenesis. To test this, the TCGA SpliceSeq database was searched and showed that exon 11 (E11) exclusion occurs frequently in several human cancers. We also found that p73a to p73g isoform switch resulting from E11 skipping occurs frequently in human prostate cancers and dog lymphomas. To determine whether p73a to p73g isoform switch plays a role in tumorigenesis, CRISPR technology was used to generate multiple cancer cell lines and a mouse model in that Trp73 E11 is deleted. Surprisingly, we found that in E11-deificient cells, p73g becomes the predominant isoform and exerts oncogenic activities by promoting cell proliferation and migration. In line with this, E11-deficient mice were more prone to obesity and B-cell lymphomas, indicating a unique role of p73g in lipid metabolism and tumorigenesis. Additionally, we found that E11- deficient mice phenocopies Trp73-deficient mice with short lifespan, infertility, and chronic inflammation. Mechanistically, Mechanistically, we showed that Leptin, a pleiotropic adipocytokine involved in energy metabolism and oncogenesis, was highly induced by p73g, necessary for p73g-mediated oncogenic activity, and associated with p73a to g isoform switch in human prostate cancer and dog lymphoma. Finally, we showed that E11-knockout promoted, whereas knockdown of p73g or Leptin suppressed, xenograft growth in mice. Our study indicates that the p73g-Leptin pathway promotes tumorigenesis and alters lipid metabolism, which may be targeted for cancer management

A PolH Transcript with a Short 3′UTR Enhances PolH Expression and Mediates Cisplatin Resistance

Platinum-based anticancer drugs are widely used as a first-line drug for cancers, such as non-small cell lung carcinoma (NSCLC) and bladder cancer. However, the efficacy is limited due to intrinsic or acquired resistance to these drugs. DNA polymerase eta (PolH, Polη) belongs to the Y-family of DNA polymerases and mediates DNA translesion synthesis, a major mechanism for DNA damage tolerance. Here, we showed that a high level of PolH is associated with cisplatin resistance in lung and bladder cancer. Consistent with this, loss of PolH markedly attenuates cisplatin resistance in both cisplatin-sensitive and cisplatin-resistant lung cancer cells. Interestingly, we found that due to the presence of multiple polyadenylation sites, alternative polyadenylation (APA) produces three major PolH transcripts with various lengths of 3'untranslated region (3'UTR; 427-/2516-/6245-nt). We showed that the short PolH transcript with 427-nt 3'UTR is responsible for high expression of PolH in various cisplatin-resistant lung and bladder cancer cell lines. Importantly, loss of the short PolH transcript significantly sensitizes cancer cells to cisplatin treatment. Moreover, we found that miR-619 selectively inhibits the ability of the long PolH transcript with 6245-nt 3'UTR to produce PolH protein and, subsequently, PolH-dependent cell growth. Together, our data suggest that PolH expression is controlled by APA and that the short PolH transcript produced by APA can escape miR-619-mediated repression and, subsequently, confers PolH-mediated cisplatin resistance. SIGNIFICANCE: A short PolH transcript produced by alternative polyadenylation escapes repression by miR-619 and confers resistance to cisplatin

Mdm2 is a target and mediator of IRP2 in cell growth control.

Iron is an essential element to all living organisms and plays a vital role in many cellular processes, such as DNA synthesis and energy production. The Mdm2 oncogene is an E3 ligase and known to promote tumor growth. However, the role of Mdm2 in iron homeostasis is not certain. Here, we showed that Mdm2 expression was increased by iron depletion but decreased by iron repletion. We also showed that Iron Regulatory Protein 2 (IRP2) mediated iron-regulated Mdm2 expression. Specifically, Mdm2 expression was increased by ectopic IRP2 but decreased by knockdown or knockout of IRP2 in human cancer cells as well as in mouse embryonic fibroblasts. In addition, we showed that IRP2-regulated Mdm2 expression was independent of tumor suppressor p53. Mechanistically, we found that IRP2 stabilized Mdm2 transcript via binding to an iron response element (IRE) in the 3'UTR of Mdm2 mRNA. Finally, we showed that Mdm2 is required for IRP2-mediated cell proliferation and Mdm2 expression is highly associated with IRP2 in both the normal and cancerous liver tissues. Together, we uncover a novel regulation of Mdm2 by IRP2 via mRNA stability and that the IRP2-Mdm2 axis may play a critical role in cell growth

TP73 Isoform-specific disruption reveals a critical role of TAp73beta in growth suppression and inflammatory response

TP73 is expressed as multiple N- and C-terminal isoforms through two separate promoters or alternative splicing. While N-terminal p73 isoforms have been well studied, very little is known about p73 C-terminal isoforms. Thus, CRISPR was used to delete TP73 Exon13 (E13-KO) to induce p73α to p73β isoform switch. We showed that E13-KO led to decreased cell proliferation and migration and sensitized cells to ferroptosis, which can be reverted by knockdown of TAp73β in E13-KO cells. To understand the biological function of p73β in vivo, we generated a mouse model in that the Trp73 E13 was deleted by CRISPR. We showed that p73α to p73β isoform switch led to increased cellular senescence in mouse embryonic fibroblasts. We also showed that E13-deficient mice exhibited shorter life span and were prone to spontaneous tumors, chronic inflammation and liver steatosis as compared to WT mice. Additionally, we found that the incidence of chronic inflammation and liver steatosis was higher in E13-deficient mice than that in Trp73-deficient mice, suggesting that p73β is a strong inducer of inflammatory response. Mechanistically, we showed that TAp73β was able to induce cysteine dioxygenase 1 (CDO-1), leading to cysteine depletion and subsequently, enhanced ferroptosis and growth suppression. Conversely, knockdown of CDO-1 was able to alleviate the growth suppression and ferroptosis in E13-KO cells. Together, our data suggest that at a physiologically relevant level, TAp73β is a strong inducer of growth suppression but insufficient to compensate for loss of TAp73α in tumor suppression due to aberrant induction of inflammatory response and liver steatosis

FDXR regulates TP73 tumor suppressor via IRP2 to modulate aging and tumor suppression.

Ferredoxin reductase (FDXR) is a mitochondrial flavoprotein that initiates electron transport from NADPH to several cytochromes P450 via two electron carriers, ferredoxin 1 (FDX1) and FDX2. FDXR is the sole ferredoxin reductase in humans and plays a critical role in steroidogenesis and biosynthesis of heme and iron-sulfur clusters. However, much less is known about the role of FDXR in cancer. Here, we show that FDXR plays a role in tumorigenesis by modulating expression of the tumor suppressor p73. By using genetically modified mouse models, we recently showed that mice deficient in either Fdxr or Trp73 had a shorter lifespan and were prone to spontaneous tumors as compared with wild-type (WT) mice. Interestingly, compound Trp73 +/- ;Fdxr +/- mice lived longer and developed fewer tumors when compared with Fdxr +/- or Trp73 +/- mice. Moreover, we found that cellular senescence was increased in Trp73 +/- and Fdxr +/- mouse embryonic fibroblasts (MEFs), which was further increased in Trp73 +/- ;Fdxr +/- MEFs, as compared with that in WT MEFs. As FDXR is regulated by p73, we examined whether there was a feedback regulation between p73 and FDXR. Indeed, we found that Trp73 expression was decreased by loss of Fdxr in MEFs and that FDXR is required for p73 expression in multiple human cancer cell lines independent of p53. Mechanistically, we found that loss of FDXR, via FDX2, increased expression of iron-binding protein 2 (IRP2), which subsequently repressed TP73 mRNA stability. We also showed that TP73 transcript contained an iron response element in its 3'UTR, which was required for IRP2 to destabilize TP73 mRNA. Together, these data reveal a novel regulation of p73 by FDXR via IRP2 and that the FDXR-p73 axis plays a critical role in aging and tumor suppression. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Small Proline-Rich Protein 2A and 2D Are Regulated by the RBM38-p73 Axis and Associated with p73-Dependent Suppression of Chronic Inflammation.

Small proline-rich protein 2A and 2D (SPRR2A and SPRR2D) provide barrier function in terminally differentiated stratified squamous epithelia through the epidermal differentiation complex. However, little is known how SPRR2A/2D expression is controlled and their role in chronic inflammation. Here, we showed that that SPRR2A/2D expression is controlled by a regulatory loop formed by RNA-binding protein RBM38 and tumor suppressor p73. Specifically, we found that SPRR2A/2D expression was induced by ectopic expression of RBM38 or p73 but suppressed by knockout of Rbm38 or p73. We also found that RBM38-mediated expression of SPRR2A/2D was p73-dependent and that induction of SPRR2A/2D during keratinocyte differentiation was dependent on both p73 and Rbm38. Additionally, we found that SPRR2A/2D expression was closely associated with p73 expression in normal and cancerous tissues. To determine the biological function of the RBM38-p73 loop potentially via SPRR2A/2D, we generated a cohort of wild-type, Rbm38-/-, Trp73+/-, and Rbm38-/-;Trp73+/- mice. We found that Rbm38-/-;Trp73+/- mice had a much shorter lifespan than that for Rbm38-/--and to a lesser extent for Trp73+/- mice-but were less prone to spontaneous tumors than Trp73+/- or Rbm38-/- mice. We also found that Rbm38-/-;Trp73+/- mice exhibited weak expression of SPRR2A/2D in multiple tissues and were susceptible to systemic chronic inflammation, suggesting that decreased SPRR2A/2D expression is likely responsible for chronic inflammation in Rbm38-/-;Trp73+/- mice, leading to a shortened lifespan. Together, our data reveal that SPRR2A/2D are novel targets of the RBM38-p73 loop and contribute to p73-dependent suppression of chronic inflammation

p73α1, a p73 C-terminal isoform, regulates tumor suppression and the inflammatory response via Notch1

p73, a p53 family member, undergoes alternative splicing at the 3′ end to produce multiple isoforms, but their expression and activity are largely unknown. Thus, CRISPR was used to knock out exon 12 (E12) in human cancer cell lines and mice, leading to isoform switch from p73α to isoform p73α1. We found that p73α1 is naturally expressed and induced by DNA damage. We also found that knockout of E12 suppresses cell growth and migration in H1299 and MIA PaCa-2 cells and promotes cellular senescence in mouse embryonic fibroblasts. Similarly, ectopic expression of p73α1 suppresses cell proliferation, whereas knockdown of p73α1 restores the cell proliferative and migratory capacities of E12−/− cells. Consistently, we found that E12+/− mice are not prone to spontaneous tumors. Instead, E12+/− mice are prone to systemic inflammation and exhibit elevated TNFα expression in inflamed tissues. Moreover, we found that Notch1, a master regulator of the inflammatory response, is regulated by p73α1 and highly expressed in E12−/− cells and inflamed E12+/− mouse tissues. Furthermore, through knockdown of p73α1 and/or Notch1 in E12−/− cells, we found that Notch1 is necessary for p73α1-mediated growth suppression. Together, these data suggest that p73α1 plays a critical role in tumor suppression and the inflammatory response via Notch1

p73α1, an Isoform of the p73 Tumor Suppressor, Modulates Lipid Metabolism and Cancer Cell Growth via Stearoyl-CoA Desaturase-1.

Altered lipid metabolism is a hallmark of cancer. p73, a p53 family member, regulates cellular processes and is expressed as multiple isoforms. However, the role of p73 in regulating lipid metabolism is not well-characterized. Previously, we found that loss of p73 exon 12 (E12) leads to an isoform switch from p73α to p73α1, the latter of which has strong tumor suppressive activity. In this study, comprehensive untargeted metabolomics was performed to determine whether p73α1 alters lipid metabolism in non-small cell lung carcinoma cells. RNA-seq and molecular biology approaches were combined to identify lipid metabolism genes altered upon loss of E12 and identify a direct target of p73α1. We found that loss of E12 leads to decreased levels of phosphatidylcholines, and this was due to decreased expression of genes involved in phosphatidylcholine synthesis. Additionally, we found that E12-knockout cells had increased levels of phosphatidylcholines containing saturated fatty acids (FAs) and decreased levels of phosphatidylcholines containing monounsaturated fatty acids (MUFAs). We then found that p73α1 inhibits cancer cell viability through direct transcriptional suppression of Stearoyl-CoA Desaturase-1 (SCD1), which converts saturated FAs to MUFAs. Finally, we showed that p73α1-mediated suppression of SCD1 leads to increased ratios of saturated FAs to MUFAs

TP73 Isoform-specific disruption reveals a critical role of TAp73beta in growth suppression and inflammatory response

Abstract TP73 is expressed as multiple N- and C-terminal isoforms through two separate promoters or alternative splicing. While N-terminal p73 isoforms have been well studied, very little is known about p73 C-terminal isoforms. Thus, CRISPR was used to delete TP73 Exon13 (E13-KO) to induce p73α to p73β isoform switch. We showed that E13-KO led to decreased cell proliferation and migration and sensitized cells to ferroptosis, which can be reverted by knockdown of TAp73β in E13-KO cells. To understand the biological function of p73β in vivo, we generated a mouse model in that the Trp73 E13 was deleted by CRISPR. We showed that p73α to p73β isoform switch led to increased cellular senescence in mouse embryonic fibroblasts. We also showed that E13-deficient mice exhibited shorter life span and were prone to spontaneous tumors, chronic inflammation and liver steatosis as compared to WT mice. Additionally, we found that the incidence of chronic inflammation and liver steatosis was higher in E13-deficient mice than that in Trp73-deficient mice, suggesting that p73β is a strong inducer of inflammatory response. Mechanistically, we showed that TAp73β was able to induce cysteine dioxygenase 1 (CDO-1), leading to cysteine depletion and subsequently, enhanced ferroptosis and growth suppression. Conversely, knockdown of CDO-1 was able to alleviate the growth suppression and ferroptosis in E13-KO cells. Together, our data suggest that at a physiologically relevant level, TAp73β is a strong inducer of growth suppression but insufficient to compensate for loss of TAp73α in tumor suppression due to aberrant induction of inflammatory response and liver steatosis