ASPP2 enhances chemotherapeutic sensitivity through the down-regulation of XIAP expression in a p53 independent manner in hepatocellular carcinoma

https://doi.org/10.1016/j.bbrc.2018.11.181Get rights and content

Highlights

  • Low ASPP2 expression fund in human tumors and facilitate a poor prognosis and metastasis.

  • Huge of HCC patients are resistant to sorafenib, and display a poor prognosis.

  • XIAP is the pivotal candidate in chemoresistance by inhibiting caspases activity.

  • ASPP2 enhances chemotherapeutic sensitivity by regulating XIAP expression and sub-localization.

Abstract

Apoptosis stimulated protein of p53-2 (ASPP2) induces the transcription of p53-targeted genes to stimulates its pro-apoptosis function. The poor chemotherapeutic sensitivity is associated with the decreased ASPP2 expression in many human cancers. Here, multiple genes real-time RT-PCR array and western blotting analysis show that ASPP2 suppress the expression of X-linked inhibitor of apoptosis protein (XIAP), determinant of chemoresistance in cancer, in hepatocellular carcinoma (HCC) in a p53-independent manner. Further experiments with ASPP2-rAd and ASPP2-Lv confirmed that ASPP2 enhanced sensitivity of sorafenib to HCC via suppressing XIAP expression. XIAP mainly found on the cytoplasm and perinuclear areas of ASPP2 over-expressed HepG2 cells, while both cytoplasm and nucleus in ASPP2 shut down HepG2 cells. The association of poor sensitivity of sorafenib and XIAP expression was also found both in ASPP2 shut down and overexpress mice, where liver tissue with decreased or increased ASPP2 displayed less or more apoptosis, respectively. Finally, ASPP2 and XIAP expression analyzed in 43 hepatocellular carcinoma tumors and 44 adjacent normal tissues from 38 hepatocellular carcinoma patients for fully understand their expression within HCC patients. Compared with the tumor tissues, ASPP2 mRNA levels were increased, and XIAP levels decreased in the adjacent normal tissues. Taken together, XIAP suppressed ASPP2 increased tumor sensitivity to chemotherapy in a p53-independent manner, which was associated with chemotherapy resistance, suggesting that p53 activation and XIAP suppression were two independent ways that ASPP2 enhance the sensitivity of chemotherapy.

Introduction

Hepatocellular carcinoma (HCC), the most common primary liver cancer with a poor prognosis, has a limited treatment option [1]. Sorafenib, a multi-targeted tyrosine kinase inhibitor, remains the major drug approved for systemic treatment in advanced HCC for pro-apoptotic activity [2]. Unfortunately, only limited patients show a regression of the tumor after the initiation of sorafenib administration [3]. A huge number of HCC patients are resistant to sorafenib which display a poor prognosis with reduced survival as their tumor progresses. At present, a number of studies and trials performed to fund the underlying mechanisms involved in the drug resistance to sorafenib in HCC. Any molecular and regents modulate the sensitivity of sorafenib bring hopes to HCC patients.

X-linked inhibitor of apoptosis protein (XIAP), baculoviral inhibitor of apoptosis repeats (BIR) in the N-terminal and RING Zn finger in the C-terminal, is the most pivotal member of the IAP family. XIAP inhibited both initiator caspases (e.g., caspase-8 and caspase-9) and effector caspases (e.g., caspases-3 and caspases-7) by an unequal mechanism. BIR2 and the linker region of BIR2 in the XIAP inhibit the activity of caspase-3 and caspase-7, and BIR3 along is enough to suppress caspase-9 [4,5]. RING Zn finger domain has an E3 ligase activity and potentiate the ubiquitination of targets [6]. Together, BIR and RING Zn finger domain in the XIAP endue the capacity for inhibiting all the caspases that induce apoptosis [7,8].

However, mechanisms that regulate XIAP expression remains a litter understood, and XIAP is high express in HCC. Though, p53 accumulation and XIAP depletion are known as a dual mechanism induce apoptosis of cancer cells [9], apoptosis induced by the down-regulation of XIAP dependent on cellular p53 status [10]. Regulators of p53 may thus also have a role in affecting cellular responses to XIAP. Among those, apoptosis-stimulating protein of p53 2(ASPP) is a potential candidate. ASPP2 promotes the pro-apoptotic transcriptional activity of p53 by promoting the binding of p53 to the downstream pro-apoptotic cofactor (e.g., PUMA, Fas/CD95 and Noxa) promoter region thought to be an effective regulator of p53 [11,12]. Though the mechanism is a litter known, low ASPP2 expressed malignant tumor does have a poor sensitivity to chemotherapy [13,14].

In this study, we confirmed that ASPP2 facilitated chemotherapy-induced cell death in vitro and vivo independent of p53 via suppressing XIAP expression. XIAP, suppress by ASPP2, facilitated sorafenib induced apoptosis in ASPP2 over-expressed mice. In HCC patients, XIAP suppressed in ASPP2 highly expressed tumor adjacent normal tissues, suggesting that the increased XIAP in ASPP2 low-expressed liver tumors might have contributed to chemoresistance following chemotherapy.

Section snippets

Cell culture

Human HCC cell lines wild-type p53 HepG2 and p53 null Hep3B were cultured in Minimum Eagle’s medium (Gibco™). ASPP2 expression was knockdown by shASPP2-lentivirus and vector-lentivirus infection. Cells infected with ASPP2-Adenovirus, and vector-Adenovirus for the over-expression of ASPP2.

Immunoblot assay

Cells harvested and lysed in a cold RIPA buffer for 30 min. The supernatant separated in SDS-PAGE gels, and separated proteins then transferred to a PVDF membrane. Membranes incubated with appropriate primary

ASPP2 suppress the expression of XIAP in HCC

Previous reports suggest that ASPP2 plays an important role in inducing tumor cells apoptosis including p53 dependent and independent pathway, but the mechanism was a litter understood. We run multiple genes qRT-PCR array with ASPP2 overexpression cells and ASPP2 shut down cells in both wtP53 cells-HepG2 and p53 null cells-Hep3B. The multiple genes qRT-PCR results showed that XIAP expression was strong influenced by ASPP2 expression following sorafenib stress for 24 h (Fig. 1 A, B, E and F).

Discussion

Various molecular mechanisms, transporter pumps, oncogenes, tumor suppressor genes, mitochondrial alteration, DNA repair, autophagy, epithelial-mesenchymal transition (EMT), cancer stemess and exosome, were associated with chemoresistance. Cross talks are essential [16,17]. p53, tumor suppressor gene, an indispensable gene in chemoresistance of advanced tumors, facilitates chemotherapeutic drug-induced apoptosis by regulating the expression of anti-apoptotic genes (MDM2, p21Waf1/Cip1 and Fas)

Funding sources

This work was supported by the National Institutes of Health grants [81272266, 81361120401]; and the Capital Health Research and Development Special grants [2018-1-1151].

Declaration of interest

The authors report no conflicts of interest.

Acknowledgments

We are very grateful to Charles D. Lopez for ASPP2+/- mice.

References (34)

  • H. Shin et al.

    Identification of ubiquitination sites on the X-linked inhibitor of apoptosis protein

    Biochem. J.

    (2003)
  • M. Holcik et al.

    XIAP: apoptotic brake and promising therapeutic target

    Apoptosis

    (2001)
  • Z. Li et al.

    Relationship of the expression levels of XIAP and p53 genes in hepatocellular carcinoma and the prognosis of patients

    Oncol Lett

    (2017)
  • E. Oda et al.

    Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis

    Science

    (2000)
  • Q.S. Tong et al.

    Downregulation of XIAP expression induces apoptosis and enhances chemotherapeutic sensitivity in human gastric cancer cells

    Cancer Gene Ther.

    (2005)
  • D. Bergamaschi et al.

    ASPP1 and ASPP2: common activators of p53 family members

    Mol. Cell Biol.

    (2004)
  • X. Liu et al.

    Exogenous p53 and ASPP2 expression enhances rAdV-TK/GCV-induced death in hepatocellular carcinoma cells lacking functional p53

    Oncotarget

    (2016)
  • Cited by (12)

    • TP53BP2: Roles in suppressing tumorigenesis and therapeutic opportunities

      2023, Genes and Diseases
      Citation Excerpt :

      Preclinical studies also showed that diethylnitrosamine (DEN) could activate NF-κB pathway in TP53BP2 deficient mice and promote the formation of mouse liver cancer model.51 Regarding chemotherapy, TP53BP2 can enhance the chemosensitivity of HCC by inhibiting the expression of X-linked inhibitors of apoptosis protein (XIAP).52 The deletion of TP53BP2 disrupts stem-like properties and chemotherapeutic resistance to HCC via Src/FAK/Snail axis.53

    • Deficiency of apoptosis-stimulating protein 2 of p53 protects mice from acute hepatic injury induced by CCl<inf>4</inf> via autophagy

      2019, Toxicology Letters
      Citation Excerpt :

      Liu et al. found that in hepatoma carcinoma cells, the combination of recombinant human adenovirus ASPP2 and oxaliplatin could significantly inhibit the growth of cancer cells by regulating ERK and STAT3 signaling molecules (Liu et al., 2017a). Studies indicated that ASPP2 may play a critical role in liver disease (Yang et al., 2019; Liu et al., 2015). However, the role of ASPP2 in acute hepatic injury remains unknown.

    View all citing articles on Scopus
    View full text