Elsevier

Biochimie

Volume 112, May 2015, Pages 85-95
Biochimie

Research paper
Impairment of the Pin1/E2F1 axis in the anti-proliferative effect of bortezomib in hepatocellular carcinoma cells

https://doi.org/10.1016/j.biochi.2015.02.015Get rights and content

Highlights

  • Bortezomib impairs HuH7 growth via the down regulation of the Pin1-E2F1 axis.

  • Pin-E2F1 impairment determines the down regulation of cyclin D1, E2F2 and cyclin A2.

  • Bortezomib effect on normal liver cells depends on the cellular proliferation rate.

Abstract

Background

The modest efficacy of available therapies for Hepatocellular carcinoma (HCC) indicates the need to develop novel therapeutic approaches. For the proteasome inhibitor Bortezomib (BZB), potentially attractive for HCC treatment, the mechanism of action is largely unknown. The BZB effect on E2Fs and the E2Fs control on the peptidylproline cis-trans isomerase (Pin1), prompted us to explore the BZB effect on the Pin1-E2F1 axis.

Methods

The tumorigenic cell line HuH7 together with the non-tumorigenic cells IHH and the human pluripotent stem cell derived hepatocytes (hPSC-H), were used as cellular models of HCC and normal liver cells, respectively.

Results

BZB reduces HuH7 growth as shown by cell counting, cell vitality test and cell cycle analysis; this is paralleled by the decrease of Pin1, E2F1, cyclin A2 and of the hyper-phosphorylated pRB. Pin1-E2F1 axis impairment justifies the anti-proliferative effect since Pin-E2F1 depletion decreases HuH7 growth while the over-expression rescues BZB-induced inhibition of proliferation. Moreover, Pin1-E2F1 promote HuH7 growth via the up-regulation of cyclin D1, cyclin E, cyclin A2, E2F2 and in part E2F3. Finally, in the control cells IHH and hPSC-H, BZB effect on cell vitality is not irrelevant, a fact correlated to the cellular proliferation rate. Thus, BZB effect on healthy liver tissue may not be entirely negligible hence caution should be exercised in its use in liver regeneration processes.

Conclusion

For the first time we prove the functional involvement of the Pin1-E2F1 axis in the anti-proliferative effect of BZB indicating Pin1-E2F as an attractive target to control HCC cell growth.

Introduction

Recent studies suggested that hepatocellular carcinoma (HCC) incidence and mortality rates have increased in the last years [1], [2], [3], [4]. Unfortunately, the efficacy of current therapeutic approaches such as liver resection, transplant and local radiofrequency ablation have limited benefits and the only approved systemic chemotherapy based on the use of the kinase inhibitor Sorafenib, can modestly prolong patient life span [5]. All together these circumstances indicate that effective anti HCC approaches are urgently needed.

The drug Bortezomib (BZB), so far indicated in the treatment of multiple myeloma and relapsed mantle cell lymphoma [6], is under evaluation as an alternative therapeutic approach for HCC [7]. BZB is a boronic acid dipeptide derivative able to inhibit the 26S proteasome [6]. The inhibition alters the physiological levels of different cellular proteins eliciting cell cycle arrest and the activation of the apoptotic program. In HCC BZB has been shown to down-regulate tumour cell proliferation by increasing the levels of the cell cycle inhibitors p27 Kip1/p21waf1/cip1 [8], [9], by reducing the levels of cyclin D1 [8], [10], by decreasing the phosphorylated form of the retinoblastoma protein pRB and the transcription factor E2F1 [8], [10].

The E2Fs family consists of transcription factors [11] encompassing anti-proliferative (E2F4–E2F8) and pro-proliferative (E2F1–E2F3) members. With regard to E2F1-E2F3, when bound to the pocket protein pRB, they are inactive and cannot trigger the transcription of pro-proliferative genes. However, in the presence of growth stimuli, pRB undergoes phosphorylation by cyclin-dependent kinases (Cdks) coupled with the cyclin partners, among which cyclin D1 is the first to be involved in the process. Upon pRB phosphorylation, the free E2Fs can activate the transcription of cell cycle-related proteins including cyclin E that bound to its Cdk further phosphorylates pRB increasing the amount of free E2Fs able to trigger cell cycle progression via the transcription of many S-phase genes, such as cyclin A2. E2F1-3 are implicated in HCC cell growth [12], [13], [14] and we have also observed that E2F1 is involved in the BZB-induced down-modulation of cell growth in some HCC cell lines [8].

Pin1, a peptidylproline cis-trans isomerase (PPIase), was recently identified as an E2F1-3 target gene [15], [16]. Pin1 catalyses the specific isomerization of Ser/Thr–Pro motifs following phosphorylation, thus inducing conformational changes capable of regulating the functions of Pin1 substrates such as p53 [17], [18], [19], p73 [20] and mutated p53 [21]. Pin1 can also control directly/indirectly the transcription level of p27Kip1 [22], p21waf1/cip1 [23] and cyclin D1 [24], [25]. Notably, the up-regulation of Pin1 and cyclin D1 is a relevant event in HCC carcinogenesis [26], [27], [28], [29], [30].

While the potential anti HHC properties of BZB are beginning to emerge [7], [8], [9], [10], [31], [32], the molecular mechanism of its action remains largely unknown. Based on the transcriptional control of E2F1-3 on Pin1 [15] and our previous observation of a BZB down-regulation of E2F1-3 [8], [31], in this work we explored the possible effect of BZB on Pin1 and on the Pin1-E2F1 axis in the HCC derived cell line HuH7.

Section snippets

Cell culture conditions

The human HCC derived cell lines HepG2, HuH7 and JHH6 were cultured as described [33], [34]; HepG2, HuH7 and JHH6 were assigned to high, intermediate and low hepatocytic differentiation grade on the basis of their capacity to synthesize albumin and ferritin [33]. HuH7 have been also analyzed by immune-cytochemical staining of cytospins from cultured cells resulting positive for the “Glypican-3” (monoclonal antibody -AcZon biotech S.r.l.), an antigen present in hepatic cancer cells. The

Phenotypic effect of BZB in HuH7

Our previous results [8] indicated that BZB is able to differentially down-regulate the proliferation of the differentiated HepG2 and of the undifferentiated JHH6 HCC cell lines, respectively. Here we extended the study to an additional HCC cell line, i.e. HuH7, with an intermediate differentiation phenotype. In HuH7 cells, BZB efficiently reduced the proteasome activity (Fig. 1A), the number of cells (Fig. 1B and Fig. S1), the cell viability (Fig. 1C), the amount of S-phase cells (Fig. 1D) and

Discussion

The phenotypic and molecular effects (Fig. 1, Fig. 2, S1 and S2) of BZB in the HuH7 cell line we used support previous observation [9], [10], [32] indicating the suitability of this cell line to further explore BZB effects in HCC cells. Here, we confirm our previous observation of a phenotypic-dependent effect of BZB in HCC cells. In HuH7, phenotypically closer to the well-differentiated HepG2, the effect of BZB is more pronounced than in the un-differentiated JHH6, as also quantified by the A

Author contribution

Dr. Farra and Dapas performed the experiments reported in Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, and Suppl. mat 3; Dr. Baiz performed the experiments reported in Fig. 1, Fig. 2 and Suppl. mat 1 and 2; Dr. Tonon performed the experiments reported in Fig. 8; Dr. Chiaretti contributed to the experiments reported in Fig. 6 and participated to the preliminary work regarding the set-up of plasmid transfection in HuH7; Prof Del Sal and Dr Rustighi in addition to provide the Ig anti Pin1, the

Conflict of interest

Finally, the authors declare that no conflict of interest exists.

Acknowledgments

This work was in part supported by the “Fondazione Cassa di Risparmio of Trieste”, by the “Fondazione Benefica Kathleen Foreman Casali of Trieste”, by the “Beneficentia Stiftung” of Vaduz Liechtenstein and by the Italian Minister of Instruction, University and Research (MIUR), PRIN 2010-11, [20109PLMH2].

References (42)

  • G. Grassi et al.

    The expression levels of the translational factors eEF1A 1/2 correlate with cell growth but not apoptosis in hepatocellular carcinoma cell lines with different differentiation grade

    Biochimie

    (2007)
  • R. Farra et al.

    Serum response factor depletion affects the proliferation of the hepatocellular carcinoma cells HepG2 and JHH6

    Biochimie

    (2010)
  • A. Jemal et al.

    Global cancer statistics

    CA Cancer J. Clin.

    (2011)
  • B. Scaggiante et al.

    Novel hepatocellular carcinoma molecules with prognostic and therapeutic potentials

    World J. Gastroenterol.

    (2014)
  • J.M. Llovet et al.

    Sorafenib in advanced hepatocellular carcinoma

    N. Engl. J. Med.

    (2008)
  • I. Zavrski et al.

    Proteasome: an emerging target for cancer therapy

    Anticancer Drugs

    (2005)
  • G.P. Kim et al.

    An international, multicenter phase II trial of bortezomib in patients with hepatocellular carcinoma

    Invest. New Drugs

    (2012)
  • I. Saeki et al.

    Bortezomib induces tumor-specific cell death and growth inhibition in hepatocellular carcinoma and improves liver fibrosis

    J. Gastroenterol.

    (2013)
  • C. Attwooll et al.

    The E2F family: specific functions and overlapping interests

    EMBO J.

    (2004)
  • F. Xiao et al.

    MicroRNA-503 inhibits the G1/S transition by downregulating cyclin D3 and E2F3 in hepatocellular carcinoma

    J. Transl. Med.

    (2013)
  • A. Xanthoulis et al.

    E2F transcription factors and digestive system malignancies: how much do we know?

    World J. Gastroenterol.

    (2013)
  • Cited by (0)

    1

    These authors contributed equally.

    View full text