Mechanisms, function and clinical applications of DNp73

Abstract

p73, has two distinct promoters, which allow the formation of two protein isoforms: full-length transactivating (TA) p73 and an N-terminally truncated p73 species (referred to as DNp73) that lacks the N-terminal transactivating domain. Although the exact cellular function of DNp73 is unclear, the high expression levels of the genes have been observed in a variety of human cancers and cancer cell lines and have been connected to pro-tumor activities. Hence the aim of this review is to summarize DNp73 expression status in cancer in the current literature. Furthermore, we also focused on recent findings of DNp73 related to the biological functions from apoptosis, chemosensitivity, radiosensitibity, differentiation, development, etc. Thus this review highlights the significance of DNp73 as a marker for disease severity in patients and as target for cancer therapy.

Introduction

p53 is one of the most frequently mutated gene in the human tumor suppressor genes. It was reported that the p53 gene played an important role in tumorigenesis progression and metastasis of human cancer. In 1997, p73 gene was found as a member of the p53 gene family^1,2 and quickly induced scholars’ tremendous interest. People thought that p73 gene belonged to the tumor suppressor gene. But with the further research of p73 gene, people have discovered that there are a lot of differences between the function of p73 gene and p53 gene, even the p73 gene function is quite contrary to p53 gene. The reason is that p73 isoforms originate from alternative splicing of the p73 genes.³ Generally p73 isoforms can be divided into two groups: transcriptionally active p73 isoforms (TAp73), which induce apoptosis and activate transcription of cell cycle regulators, and N-terminally truncated variants (referred to as DNp73), which lack the N-terminal transactivation domain and inhibit TAp73 and p53 activity.^3-7 While much is known about the TAp73 expression in cancer, the effect of DNp73 status on other biological function is less well understood. The aim of this review is to summarize DNp73 expression status in cancer in the current literature. We also review biological functions of DNp73 from cell apoptosis, chemosensitivity, radiosensitivity, differentiation, development, etc, thus highlighting the significance of DNp73 as a marker for disease severity in patients and as target for cancer therapy.

Structure of DNp73

p73 has been identified as a structural and functional homolog of the tumor suppressor protein p53 based on sequence conservation of the transactivation (TA), DNA binding (DBD) and oligomerization domains (OD) of these proteins.^1,8 The p73 gene possesses an extrinsic P1 promoter and an intrinsic P2 promoter, resulting in TAp73 and DNp73 isoforms, respectively (Fig. 1A). The P1 transcripts excluding TAp73 are referred to as the ΔTAp73 isoforms and exhibit broadly similar functions.^9,10 Similar to the ΔNp73 isoforms, ΔTAp73 isoforms can also be C-terminally spliced, generating at least eight different splice variants.¹¹ p73 is expressed in multiple variants arising from alternative splicing of the primary p73 transcript, including the C-terminal isoforms and at least seven different transcripts. p73α is the longest form, containing a sterile a motif domain (SAM) in the extreme COOH-terminal region. All other isoforms are rearranged in the COOH-tail and lack the SAM domain (Fig. 1B). These splicing variants are expressed differently in normal human tissues and cell lines. Besides TAp73, four different NH2 terminally truncated isoforms, ΔNp73, ΔN′p73, ΔEx2p73 and ΔEx2/3p73, have been found in human cancers and cancer cell lines (Fig. 1B). Each one lacks all or most of the transactivating domain, so they are collectively called DNp73 (Fig. 1B). DNp73 products are generated either through alternative exon splicing of the P1 promoter (producing ΔN′p73, ΔEx2p73 and ΔEx2/3p73), or by use of the P2 promoter in intron 3, producing ΔNp73. The ΔEx2p73 and ΔEx2/3p73 isoforms lack exon 2 and exon 2/3, respectively (Fig. 1B and C). The transcripts ΔNp73 and D′Np73 encode the same protein product (Fig. 1C).

Figure 1. Gene structure and alternative transcripts of human TP73. (A) Domain structure of p73. (B) Pattern of NH₂-terminal truncated isoforms (DNp73, DN’p73, Ex2p73 and Ex2/3p73). Exons are depicted as purple boxes. The two promoter regions are displayed as gray boxes (P1, P2). (C) NH2-terminal transcript variations. The exon structure of the individual mRNAs and position of primers used for specific isoform amplification is marked by arrows, and the encoded proteins are displayed.

DNp73 Isoforms Expression in Cancer

Extensive studies reported the N-terminally truncated p73 variants in human cancers,^11,12 using quantitative real-time PCR with isoform-specific primers to distinguish between the various DNp73 and TAp73.¹³ This is basically due to the lack of available high-affinity, isoform-specific antibodies. With the developing of technology, antibodies generated against an exon 3′-specific epitope to detect the ΔNp73 protein are commercially available. Specific primers and this kind of antibodies provide emerging evidence that N-terminally truncated p73 isoforms act as a biologically relevant oncogene in primary human cancers. Additionally, isoform-specific knockout (KO) model, RNAi techonology and transgenic mice are powerful tools to study the role of DNp73. DNp73 were found frequently upregulated in many other human cancers (summarized in Table 1). ΔNp73 is upregulated in a number of primary tumors including breast,^14,15 colon,^14,16 lung,^17,18 ovary,^19-21 cervix,^15,22 thyroid,^23-25 acute myeloid leukemia,²⁶ neuroblastoma,^27-29 etc. (Table 1). For example, a significant increase of DNp73 was seen in 20 of 33 carcinomas and 17 of 24 benign prostate hyperplasia tissues but in none of the normal samples.³⁰ These data suggest a potential role for DNp73 in prostate cancer progression. Accordingly, the recent research showed how this effect of ΔNp73 could be a contributing factor in cancer progression.³¹ The upregulation of the ΔNp73 protein is also significantly associated with poor patient survival in gastric, gastresophageal junction and esophageal tumor.³² Overexpression of ΔEx2/3p73 and ΔNp73 was associated with advanced pathologic tumor stages of colon and breast cancers.¹⁶ In addition, Uramoto et al. detected ΔNp73 expression in lung cancer by using immunohistochemical (IHC) staining and evaluated the relationship between ΔNp73 expression levels of tumors and the prognosis of the patients.¹⁸ The results indicated that expression of ΔNp73 may be a useful marker for predicting poor prognosis of patients who underwent resection of lung cancer.¹⁸ Soon after, Liu et al. showed that in cervical cancer, overexpression of ΔNp73 was a potential indicator in predicting the risk of the disease recurrence, as it was observed to be significantly higher in patients with recurrence of disease (70.8%).²² Moreover, several reports indicated considerable upregulation of DNp73 in hepatocellular carcinoma (HCC), which correlates with reduced survival of patients, but little is known about the functional significance of DNp73 to tumorigenesis in vivo. Just then Tannapfel et al. showed that transgenic mice displayed increased proliferation of hepatocytes, with acinar disorganization and the appearance of pre-neoplastic nodules (adenomas) that in the 83% of cases evolved in HCC.³³ With respect to an equally important role for DNp73 in the tumorigenic process, Emmrich et al. 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 exon3B unique for ΔN' and ΔN.³⁴ This data supported the application of NH2-truncated p73 inhibitors as valuable tools to delineate their biological role in human cancers and as anticancer agents. Indeed, many in vitro and in vivo evidences demonstrate that the DNp73 have an oncogenic role.⁴ However, a body of research has suggested that ΔNp73 overexpression alone does not confer any growth advantage to tumor cells.^5,35 Thus, this observation lead to the assumption that ΔNp73 was not an oncogene; nevertheless, with genotoxic stimuli or acquisition of a Ras mutation, high ΔNp73 expression does lead to increased cell survival.⁵ This contrary function may be explained by the following reasons: (1) The ratio between TAp73 and DNp73 dictates the cellular response;^7,15,36,37 (2) ΔNp73 upregulation occurs regardless of p53 mutation status, but the consequences of this depend on the cell type;^5,38 (3) A comprehensive understanding of TAp73 and DNp73 post-translational modifications will be tremendously useful for understanding the roles in cancer.³⁷

Table 1. DNp73 expression in different human cancers.

Organ/tissue/cell line	Isoform expression	Method of analysis	References
Ovarian cancer	ΔNp73↑	IHC; RT-PCR	15 and 21
	ΔN′p73↑;ΔNp73↑ only in small number of tumors	Real-time PCR	19
	ΔN′p73↑	RT-PCR	76
Lung cancer	ΔNp73↑	RT-PCR; IHC; Antisense Oligonucleotide (ASO)	17, 18, 33 and 77
Non-small cell lung carcinomas (NSCLCs)	ΔΝp73↑	RT-PCR; IHC	2, 78 and 79
Cervix carcinoma	ΔNp73↑	IHC; RT-PCR	15, 22 and 48
Thyroid carcinoma	ΔNp73↑	RT-PCR; IHC	24 and 25
		Transfection	23
Breast cancer	ΔEx2/3p73↑;ΔNp73↑;ΔEx2p73↑	RT-PCR, IHC	14 and 15
Hepatocellular carcinoma (HCC)	p7Δex2↑;p73Δex2/3↑; ΔN’-p73↑	Real-time PCR	80
	ΔEx2p73↑		36
	ΔNp73↑	Western blot analysis; IHC	81
	DNp73↑	RT-PCR; western blotting	56
	ΔNp73β↑	Transfection	57
Neuroblastoma	ΔNp73↑	RT-PCR; Immunoblotting	28, 29
	ΔNp73↓	Antisense	27
Colon cancer	ΔEx2/3p73↑;ΔNp73↑;ΔEx2p73↑	RT-PCR, IHC	14
	ΔEx2/3p73↑; ΔNp73↑	Real-time RT-PCR	16
Gynecological cancer	ΔNp73↑; ΔN'p73↑	Real-time RT-PCR	38
	ΔNp73↑	RT-PCR	15
	ΔEx2p73↑		82
Prostate carcinoma	ΔNp73↑	RT-PCR	83
Acute myeloid leukemia	ΔNp73↑	RT-PCR; western blotting	26
Non-Hodgkin's lymphomas	ΔNp73↑	Real-time PCR	84
Glioma	p73Δex2/3↑;ΔEx2p73↑	Real-time PCR	85
Medulloblastoma	ΔNp73↑	IF; IHC; Real-time PCR western blotting	86–88
	ΔEx2/3p73↑,ΔNp73↑;ΔEx2p73↑	RT-PCR	88
Ameloblastoma	ΔNp73↑	RT-PCR; IHC	89
Osteogenic Sarcoma	ΔNp73↑	RT-PCR;Western blotting	90
Chronic B-cell lymphocytic leukemia	ΔNp73↑	RT-PCR; western blotting	91
Head and neck squamous cell cancer (HNSCC)	ΔNp73↑	RT-PCR; Real-time PCR	92
Rhabdomyosarcoma	ΔEx2p73↑;ΔNp73↑	RT–PCR	72
Gastric and esophageal tumors	ΔNp73↑	RT-PCR; western blotting	32

DNp73 and Apoptosis

DNA damage can also induce apoptotic cell death in order to preserve genome integrityl.³⁹ Unchecked DNA damage can lead to mutations, translocations and abnormal recombination events during S phase and also chromosome breakage and loss during mitosis. Failure to monitor or signal damaged DNA is a hallmark of cancer cells.⁴⁰ The choice between cell cycle arrest and apoptosis is influenced by both the quality and the strength of the DNA damage imposed to the cell. p73, like its homolog, the tumor suppressor p53, may act as transcription factors with common target sequences whose transcriptional activation can lead to cell cycle arrest and apoptosis.^6,27,41,42 This property is important for the involvement of p73 in cancer development and therapy. However, in contrast with p53, while the transactivation-proficient TAp73 shows pro-apoptotic effects, the DNp73 has an anti-apoptotic function. ΔNp73 has been linked to the ability to act as dominant-negative of the TA isoforms as well as p53. Indeed ΔNp73 can repress the transcriptional activity of TAp73 through the formation of inactive oligomers as well as competing for DNA binding while it antagonizes p53.⁴³ Interestingly, both p53 and TAp73 induce ΔNp73, creating an auto-regulatory feedback loop. By interfering with p53 and TAp73, the DNp73 isoforms inhibit apoptosis and promote cell cycle progression.^31,44 For example, DNp73 is expressed in vivo in the developing brain, where it counteracts p53-dependent apoptosis.⁴⁵ In the only study published so far with a DNp73-specific antisense oligonucleotide, downregulation of the protein induced apoptosis in colon carcinoma cells, either upon expression of exogenous p53 or induction of DNA damage.¹⁵ Up to now, 7001 is a highly effective DNp73 antisense molecule, and silencing of these transcripts induces apoptosis in neuroblastoma cells.²⁷ Abrogation of DNp73 expression by specific siRNA led to a strong potentiation of the spontaneous apoptosis of C2C12 myoblasts induced to differentiate.⁴⁶ In cultured sympathetic neurons, overexpression of ΔNp73 inhibits apoptosis induced by nerve growth factor withdrawal or p53 overexpression.^45,47 Thus, ΔNp73 is an essential anti-apoptotic protein in neurons, serving to counteract the pro-apoptotic function of p53. In addition, in human cervix carcinoma HeLa cells, downregulated ΔNp73 expression might play a critical role in promoting cycle arrest and apoptosis in cancer cells.^48. Furthermore, ΔNp73 KO mouse shows that cells from ΔNp73 (−/−) mice are sensitized to DNA-damaging agents and show an increase in p53-dependent apoptosis.⁴⁹ The recent study shows that DNp73 acts as an oncogene to suppress p53- and p73-induced apoptosis.⁵⁰ Indeed, DNp73 can inhibit apoptosis and promote cell cycle progression. While, Taebunpakul et al. suggested a novel mechanism of apoptin-induced apoptosis through increased TAp73 stability and induction of PIR2, resulting in the degradation of ∆Np73 and activation of pro-apoptotic targets such as PUMA causing cancer cell death.⁵¹ Interestingly, in response to DNA damage, ΔNp73 isoforms are degraded, thus allowing the TA forms to exert their effect. Additionally, Ory et al. uncovered a mechanism which maintains p63/p73 homeostasis within the epithelium through direct transcriptional regulation of microRNAs.⁵² The next year, Alla et al. reported that metastatic cells could be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and reduction of tumor growth in vivo.⁴² Soon, Vera et al. reported that downregulation of miR-205 could be mediated by an imbalance in the p73/DNp73 ratio or by dysregulation of other cancer-related regulators of miR-205 expression like TGFβ-1 or TWIST1.⁵³ In addition, it has been shown that ΔNp73 can inhibit neuronal cell death through direct binding to the c-Jun N-terminal kinase (JNK), thus reducing Bim-EL expression and therefore the apoptotic mitochondrial pathway.⁵⁴ Therefore, DNp73 has an anti-apoptotic function can be designed as cancer therapy target.

DNp73 and Chemosensitivity

Currently, the use of chemotherapeutics remains the predominant option for cancer therapy. However, one of the major obstacles for successful cancer therapy using these chemotherapeutics is that patients often do not respond or eventually develop resistance after initial treatment.⁵⁵ Therefore identification of genes involved in chemotherapeutic response is critical for predicting tumor response and treating drug-resistant cancer patients. Since p53 and TAp73-mediated apoptosis can be augmented by various cancer chemotherapeutic agents, it has been hypothesized that the status of the endogenous p53 and TAp73 genes in cancer cells are key determinants in the outcome of cancer therapy.¹³ Whereas DNp73 are clearly capable of inhibiting drug-induced apoptosis in cancer cells that retain wild-type p53 and/or TAp73, thereby conferring chemoresistance,^36,55,56 Zaika et al., reported that downregulation of endogenous ΔNp73 levels by antisense methods enhances p53/TAp73-mediated apoptosis in cancer cells in response to chemotherapy.¹⁵ Schuster et al. reported that ΔNp73 isoforms repress apoptosis-related genes of the extrinsic and intrinsic apoptosis signaling pathways, thereby contributing to chemoresistance.⁵⁷ Moreover, increased ΔNp73 expression in tumors has been correlated with chemotherapeutic failure. Wilhelm et al. generated cells from ΔNp73^−/− mice that are sensitized to DNA-damaging agents and show an increase in p53-dependent apoptosis.⁴⁹ ΔNp73 localizes directly to the site of DNA damage, can interact with the DNA damage sensor protein 53BP1 and inhibits ATM activation and subsequent p53 phosphorylation.⁴⁹ This novel finding may explain why human tumors with high levels of ΔNp73 expression show enhanced resistance to chemotherapy. Furthermore, it has also been shown both in vitro and in clinical cancers that mutant ΔNp73 can directly bind and inhibit TAp73, resulting in reduced transcriptional activation of pro-apoptotic genes Bax, PUMA and p53AIP1, ultimately rendering cancer cells and patients resistant to chemotherapeutic drug treatment.^55,58,59 Soon after, Alla et al. provided evidence that miR-205 mediates the DNA damage response of p73, whereas its repression by rather low DNp73 levels was crucial for E2F1 to induce chemoresistance.⁴² It was reported that inducible chemoresistance mediated by this miR- DNp73 mechanism might be an attractive target for therapeutic intervention.⁵³ While Conforti et al. showed that the relative ratio of different p73 isoforms was critical for the cellular response to a chemotherapeutic agent.⁷ In a word, the expressions of the isoform (ΔNp73) might be potential markers for predicting the prognosis and sensitivity to chemotherapy in patients.

DNp73 and Radiosensitivity

The success of treatment of cancer patients by radiotherapy largely depends on tumor radiosensitivity. Several molecular factors that determine the sensitivity of tumor cells to ionizing radiation showed alternative transcription initiation as a response to irradiation and have been identified during the last couple of years.^40,60 One of them is DNp73. High expression levels of ΔNp73 have been shown to strongly correlate with poor survival of cancer patients, and ΔNp73-positive tumors show a reduced response to chemotherapy and irradiation.⁶¹ Liu et al. suggested that p73a is an important determinant of cellular radiosensitivity in the p53-impaired cervical cancer cells, whereas upregulation of ΔNp73 in cervical cancers was detected mainly in radioresistant cases.^22,62 Our findings have shown that there is a differential ΔNp73 expression in response to different LET radiations, and downregulated ΔNp73 expression might play a critical role in sensitivity of tumor cells.⁴⁸ Finally, similar findings have been reported that the anti-apoptotic ΔNp73 decreased in colon cancer cell lines (KM12C) exposed to γ-irradiation.⁶³ Thus, these findings suggested that DNp73 expression was related to the radiosensitivity of cancer cells and may play an important role in the regulation of cellular radiosensitivity.

DNp73: Differentiation and Development

Recent studies show that the p73 is critical in several types of cellular differentiation, such as neuronal differentiation, myogenic or epithelial differentiation.^{13, 64-66} ΔNp73 has an important counterbalancing role, promoting the survival of mature neurons.⁶⁷ Indeed, direct functional studies showed that ΔNp73 can function as a prosurvival factor of differentiated neurons.^45,67 Based on a report by Zhang et al., ΔNp73 modulates nerve growth factor-mediated neuronal differentiation through repression of TrkA.⁶⁸ Overexpression of ΔNp73 was found to inhibit apoptosis induced by nerve growth factor withdrawal or p53 overexpression.^45,54,69 Interestingly, ΔNp73 inhibits all forms of oligodendrocyte precursor cells differentiation by blocking TAp73 isoforms.⁷⁰ During nephrogenesis, ΔNp73 is expressed preferentially in proliferating nephron precursors.⁶⁴ The prosurvival role of ΔNp73 for differentiated mature neurons was recently confirmed in vivo by a ΔNp73 isoform-specific KO model.⁴⁷ A recently reported ΔNp73 KO mouse shows a similarly subtle phenotype with late signs of neuronal loss and neurodegeneration.⁴⁹ Mice deficient in ΔNp73 displayed neuronal pathologies, including hydrocephalus and hippocampal dysgenesis.⁷¹ Additionally, ΔNp73 plays an important role in differentiation control, preventing myogenic differentiation and cooperating oncogenes to transform myoblasts to tumorigenicity.⁷² Meanwhile, some research showed that DNp73α is expressed in proliferating C2C12 myoblasts, rapidly accumulates in differentiating myocytes and remains elevated in C2C12 myotubes.⁴⁶ Moreover, a recent study showed that p73 gene is developmentally regulated during kidney organogenesis, and that the spatiotemporal switch from ΔNp73 to TAp73 may play an important role in the terminal differentiation program of the developing nephron.⁷³ Interestingly, the recent study also showed that DNp73 improves generation efficiency of human induced pluripotent stem cells.⁵⁰ Thus, such observations support a model whereby ΔNp73 might regulate cellular differentiation or development, at least in part by antagonizing other p53 family members.

DNp73: Other Functions

Interestingly, analysis of TA/ΔN ratio in some mouse tissues confirmed that higher relative levels of ΔNp73 was the main isoform detected in uterus, salivary gland and tongue.⁷ Finally, comparable levels of TA and ΔNp73 mRNAs were expressed in skin, brain, colon and ovary. It is suggesting that ΔNp73 may have a physiological role in these tissues. Additionally, a new study reveals a surprising neuroprotective role for DNp73. Furthermore, aged mice with reduced ΔNp73 levels exhibit tau pathology and cognitive deficits, and ΔNp73 reduction in mice with amyloid pathology causes extensive tangle formation and neuron death.⁷⁴ Hence these findings provide a novel animal model of Alzheimer disease and a potential therapeutic role for ΔNp73 inducers. Soon after, the study reported the ability for p73 to regulate tyrosine hydroxylase and, by doing this, to protect against events that can lead to Parkinson disease.⁷⁵ Following this line, it would be important to study the link between individiual DNp73 and Parkinson disease.

Conclusions and Perspective

The TAp73 isoforms or p53 are regarded as tumor suppressor genes. ΔNp73 isoforms lack the N-terminal transactivation domain, hence cannot induce the expression of pro-apoptotic genes, but still can oligomerize with TAp73 or p53 to block their transcriptional activities. The anti-apoptotic DNp73-isoforms act antagonistically with possible oncogenic activity. Last but not least, a further level of diversity is generated by the complexity of differential splicing at the p73 C terminus. The transcription factor p73 can be expressed as at least 24 different isoforms with pro- or anti-apoptotic functions. For future research it has to be kept in mind that there is not only one p73 isoform. Despite the recent advances in understanding the roles of DNp73, there are still many outstanding questions. What are the unique functions of DNp73? How DNp73 is regulated by the p53 and TAp73 variants in the cancer cell or normal cell? What are the patterns of p73 isoform expression during both normal development and tumorigenesis? How is DNp73 regulated in the post-translational modifications? Overall, it becomes increasingly clear that DNp73 has played a critical role in tumor cell apoptosis, chemotherapy, radiotherapy, differenciation and development. DNp73 is not just a relative of p53, but has created a new identity on its own.

Glossary

Abbreviations:

DBD

DNA binding

DNp73

N-terminally truncated p73 species

KO

knockout

RT-PCR

reverse transcriptase polymerase chain reaction

qRTPCR

quantitative real-time polymerase chain reaction

OD

oligomerization domains

TA

transactivation

TAp73

transcriptionally active p73 isoforms

SAM

sterile a motif domain

HCC

hepatocellular carcinoma

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.