Possible Role of IRS-4 in the Origin of Multifocal Hepatocellular Carcinoma

Abstract

New evidence suggests that insulin receptor substrate 4 (IRS-4) may play an important role in the promotion of tumoral growth. In this investigation, we have evaluated the role of IRS-4 in a pilot study performed on patients with liver cancer. We used immunohistochemistry to examine IRS-4 expression in biopsies of tumoral tissue from a cohort of 31 patient suffering of hepatocellular carcinoma (HCC). We simultaneously analyzed the expression of the cancer biomarkers PCNA, Ki-67, and pH3 in the same tissue samples. The in vitro analysis was conducted by studying the behavior of HepG2 cells following IRS-4 overexpression/silencing. IRS-4 was expressed mainly in the nuclei of tumoral cells from HCC patients. In contrast, in healthy cells involved in portal triads, canaliculi, and parenchymal tissue, IRS-4 was observed in the cytosol and the membrane. Nuclear IRS-4 in the tumoral region was found in 69.9 ± 3.2%, whereas in the surrounding healthy hepatocytes, nuclear IRS-4 was rarely observed. The percentage of tumoral cells that exhibited nuclear PCNA and Ki-67 were 52.1 ± 7%, 6.1 ± 1.1% and 1.3 ± 0.2%, respectively. Furthermore, we observed a significant positive linear correlation between nuclear IRS-4 and PCNA (r = 0.989; p < 0.001). However, when we correlated the nuclear expression of IRS-4 and Ki-67, we observed a significant positive curvilinear correlation (r = 0.758; p < 0.010). This allowed us to define two populations, (IRS-4 + Ki-67 ≤ 69%) and (IRS-4 + Ki-67 > 70%). The population with lower levels of IRS-4 and Ki-67 had a higher risk of suffering from multifocal liver cancer (OR = 16.66; CI = 1.68–164.8 (95%); p < 0.05). Immunoblot analyses showed that IRS-4 in normal human liver biopsies was lower than in HepG2, Huh7, and Chang cells. Treatment of HepG2 with IGF-1 and EGF induced IRS-4 translocation to the nucleus. Regulation of IRS-4 levels via HepG2 transfection experiments revealed the protein’s role in proliferation, cell migration, and cell-collagen adhesion. Nuclear IRS-4 is increased in the tumoral region of HCC. IRS-4 and Ki-67 levels are significantly correlated with the presence of multifocal HCC. Moreover, upregulation of IRS-4 in HepG2 cells induced proliferation by a β-catenin/Rb/cyclin D mechanism, whereas downregulation of IRS-4 caused a loss in cellular polarity and in its adherence to collagen as well as a gain in migratory and invasive capacities, probably via an integrin α2 and focal adhesion cascade (FAK) mechanism.

Actinomycin D Arrests Cell Cycle of Hepatocellular Carcinoma Cell Lines and Induces p53-Dependent Cell Death: A Study of the Molecular Mechanism Involved in the Protective Effect of IRS-4

“…Previous research has demonstrated that the stable transfection of pcDNA (IRS-4) increased IRS-4 protein levels in HepG2 cells (C1 line) in comparison to a control group (pcDNA empty vector) [ 26 ]. In Figure 5 A, we show the levels of IRS-4 mRNA in both C1 and control cells (pcDNA).…”

“…Stable transfectants were obtained after selection with G418 for different periods of time. Several colonies overexpressing IRS-4 were obtained [ 26 ]. For further studies, the colony C1 was selected.…”

“…Additionally, IRS-4 is not able to be inhibited by tyrosine phosphatase SHP-2; thus, it can constitutively activate the AKT pathway [ 18 ]. Interestingly, IRS-4 is overexpressed in both benign proliferative lesions, such as uterine leiomyomas [ 19 ] and subungual exostosis [ 20 ], and malignant diseases, such as breast cancer [ 21 ], leukemia [ 22 ], lung cancer [ 23 ], colorectal cancer [ 24 , 25 ], and liver cancer [ 26 ].…”

Patients with Invasive Lobular Carcinoma Show a Significant Increase in IRS-4 Expression Compared to Infiltrative Ductal Carcinoma—A Histopathological Study

“…Insulin receptor substrate 4 (IRS-4) is a relevant molecule altered in different types of malignancies like BC. It seems that this molecule promotes tumoral proliferation and therapy resistance due to the alteration of different molecular pathways [13][14][15]. Furthermore, previous studies have found that an overexpression of this component is frequently related to a poorer prognosis [16].…”

Papel de IRS2 en la regulación de la comunicación a través de FGFs en el nicho de células progenitoras hepáticas

“…La expresión de IRS4 presenta un patrón más restringido encontrándose principalmente en cerebro, riñones, timo e hígado. En hígado, la expresión de Irs4 aumenta junto con la de Irs2 durante la regeneración hepática [52] y se ha visto involucrado con la progresión del carcinoma hepatocelular (HCC) [53]. El ratón con deleción de Irs4 (Irs4-/-) presenta únicamente un retraso moderado del crecimiento y una intolerancia leve a la glucosa [54].…”