Contribution of Galectin-1 to hepatocellular carcinoma cell drug resistance.

CARABIAS, P; BACIGALUPO, ML; RUBIN A; SAFFIOTI, N; ELOLA, MT; LANARI C; ROSSI JP; CARLOTA WOLFENSTEIN-TODEL; ROJAS P; GABRIEL A. RABINOVICH; ESPELT M.V; TRONCOSO, MF

Congreso; SAFIS-SAIC; 2018

Hepatocellular carcinoma (HCC), the most common type of liver cancer, has a poor prognosis accounting for the second leading cause of cancer-related deaths [1]. HCC usually develops from liver fibrosis and cirrhosis irrespective of the etiology of the liver disease. The most frequent risk factors for developing HCC are chronic viral hepatitis B or C virus infection, excessive alcohol consumption and nonalcoholic steatohepatitis (NASH) [2]. Regarding treatment, when HCC is diagnosed at early stages partial hepatectomy is the therapeutic choice, however in most patients the tumor is not detected promptly. In intermediate stages, transarterial chemoembolization with doxorubicin (DOX), a topoisomerase II inhibitor that induces apoptosis, is the treatment of choice. Recent efforts allowed the development of targeted molecular therapy with sorafenib, a multikinase inhibitor with antiproliferative and antiangiogenic properties that slightly improve survival in patients with advanced HCC [3]. Nevertheless, HCC still has a high mortality rate, largely because of high recurrence rate, metastasis and resistance to therapy. HCC is characterized by a high resistance to apoptosis and chemotherapy. Apoptosis or programmed cell death is a recognized mechanism that acts as a natural barrier to cancer development. However one of the hallmarks of cancer is the ability to resist cell death, either induced by physiological stress due to the tumorigenesis progress, induced by cytotoxic drugs or by molecular targeted therapies. A diverse range of molecular mechanisms have been implicated in drug resistance; including mutations in tumor suppressor genes like p53, activation of prosurvival signaling, mutations in drug targets and decrease in intracellular drug concentration by alterations in drug metabolism and drug efflux. Several cell membrane transport proteins have been linked with drug efflux and an increased resistance to commonly used chemotherapeutics. ATP-binding cassette (ABC) transporter family includes ATP-dependent pumps that cause the efflux of various hydrophobic compounds and xenobiotics such as chemotherapeutics drugs. The most studied and relevant protein involved in multi-drug resistance (MDR) is P-glycoprotein(P-gp; MDR1). P-gp is overexpressed in many tumors (causing intrinsic multidrug resistance) and its expression can be induced by chemotherapy (causing acquired multidrug resistance) [4]. In HCC, P-gp overexpression is associated with chemotherapeutic failure [5]. In addition, it was shown that prolonged treatment with chemotherapeutic drugs can induce an increase in P-gp expression in HCC cells [6]. Galectin-1 (Gal1) is a glycan-binding protein with affinity for β-galactoside-containing glycoconjugates. Gal1 has important roles in cancer biology such as tumor transformation, growth, invasion and metastasis, angiogenesis and immune-tumor escape [7] [8] [9]. In normal adult liver, Gal1 levels are undetectable [10], whereas in HCC it is over-expressed due to the increase in the levels of its mRNA produced by hypo-methylation of the LGALS1 gene promoter [11]. Gal1 overexpression in HCC correlates with tumor aggressiveness, metastasis, and enhanced risk of post partial-hepatectomy recurrence [12] [13]. Thus, Gal1 is considered as a marker of HCC poor prognosis [14]. Previously, we described that Gal1 promotes HCC cell adhesion and polarization favoring tumor growth and metastasis in vivo [15]. Further, we demonstrated that Gal1 overexpression induces epithelial-mesenchymal transition (EMT) in HepG2 HCC cells, a key process during cancer invasion [16]. Besides, we identified Gal1 as a modulator of HepG2 cell proliferation and sensitivity to transforming growth factor β1 (TGF-β1)-induced growth inhibition [17].Recent studies have shed light on Gal1 role in HCC chemoresistance. It was shown that Gal1 induces HCC resistance to sorafenib by activating FAK/PI3K/AKT signaling pathway [18]. Interestingly, increased Gal1 expression in HCC patients? serum was associated with poor treatment efficacy of sorafenib [19]. Additionally, Su et al. demonstrated that Gal1-induced autophagy is a mechanism involved in HCC resistance to cisplastin, another chemotherapeutic drug [20]. However, these are the only reports linking Gal1 with HCC resistance to chemotherapy and the precise mechanisms underlying this effect remain unclear. The aim of this work was to investigate the molecular basis of Gal1-mediated chemoresistance in HCC cells. We used the well-differentiated and low invasive human HCC cell line, HepG2, and performed ?gain-of-function? experiments by transfecting cells with Gal1 cDNA constructs (HepG2-Gal1 cells) or expression vector alone as control (HepG2-Mock cells). ?Loss-of-function? experiments were performed by transfecting cells with a pool of three target-specific Gal1 shRNAs (HepG2-shGal1 cells) or non-targeting scramble shRNAs as a control (HepG2-shScr cells) (Figure 1A). First, to investigate the involvement of Gal1 in HCC chemoresistance in vivo, we evaluated whether the overexpression of this lectin in HepG2 cells could generate DOX-resistant tumors. HepG2-Mock or HepG2-Gal1 (5x106) cells were subcutaneously inoculated into immunodeficient NOD/SCID mice. Tumor growth was measured every 3?4 days. When the tumors reached approximately 0.06 cm3, mice were assigned (five animals per group) to control (saline solution i.v.) or DOX treatment (4.5 mg/kg i.v. once a week for 3 weeks). At the end of the experiment, mice were sacrificed and tumor samples were collected for protein expression analysis by Western blot. Mean tumor volume was calculated and plotted versus time to determine response to therapy. We observed a significant increase in the volume of HepG2-Gal1-derived tumors treated with DOX compared with HepG2-Mock-derived treated tumors (1.39±0.38 cm3 vs 0.43±0.03 cm3 at the end of the experiment, p