Shift in VEGFA isoform balance towards more angiogenic variants is associated with tumor stage and differentiation of human hepatocellular carcinoma.

Mikhail S Chesnokov,Olga V Morozova,Leonid M Dyakov,Polina A Khesina,Nikolay E Kudashkin,Yuri I Patyutko,Inna F Kustova,Ekaterina A Moroz,Natalia L Lazarevich,Nikolai S Mugue,Darya A Shavochkina

doi:10.7717/peerj.4915

Abstract

BackgroundHepatocellular carcinoma (HCC) is the most common and aggressive type of malignant liver tumor. HCC progression depends significantly on its vascularization and formation of new blood vessels. Vascular endothelial growth factor A (VEGFA) is a crucial regulator of tumor vascularization and components of VEGF-induced cell signaling pathways are important targets of therapeutical drugs that demonstrated the highest efficiency in case of advanced HCC (sorafenib and regorafenib). VEGFA is expressed as a set of isoforms with different functional properties, thus VEGFA isoform expression pattern may affect tumor sensitivity to anti-angiogenic drugs. However, information about VEGFA isoforms expression in HCC is still incomplete and contradictory. The present study aims to quantitatively investigate VEGFA isoform expression aberrations in HCC tissue.MethodsA total of 50 pairs of HCC and non-tumor tissue samples were used to evaluate the VEGFA isoform spectrum using RT-PCR and quantitatively estimate changes in isoform expression using RT-qPCR. Correlations between these changes and tumor clinicopathological characteristics were analyzed.ResultsWe identified VEGFA-189, VEGFA-165, and VEGFA-121 as predominant isoforms in liver tissue. Anti-angiogenic VEGFA-xxxb variants constituted no more than 5% of all mature VEGFA transcripts detected and their expression was not changed significantly in HCC tissue. We demonstrated for the first time that the least active variant VEGFA-189 is frequently repressed in HCC (p < 0.001), while no uniform changes were detected for potent angiogenesis stimulators VEGFA-165 and VEGFA-121. Isoform balance in HCC shifts from VEGFA-189 towards VEGFA-165 or VEGFA-121 in the majority of cases (p < 0.001). Changes in fractions, but not expression levels, of VEGFA-189 (decrease) and VEGFA-121 (increase) correlated with advanced Tumor-Node-Metastasis (TNM) and Barcelona Clinic Liver Cancer (BCLC) tumor stages (p < 0.05), VEGFA-189 fraction reduction was also associated with poor tumor differentiation (p < 0.05).DiscussionA distinct shift in VEGFA isoform balance towards more pro-angiogenic variants occurs in HCC tissue and may modulate overall impact of VEGFA signaling. We suppose that the ratio between VEGFA isoforms is an important parameter governing HCC angiogenesis that may affect HCC progression and be used for optimizing the strategy of HCC therapy by predicting the response to anti-angiogenic drugs.

Highlights

Hepatocellular carcinoma (HCC) is the most common and aggressive form of primary liver tumor and ranks second place in cancer-related mortality rates (Llovet et al, 2016)
Vascular endothelial growth factor A (VEGFA)-189, VEGFA-165, and VEGFA-121 are major VEGFA isoforms expressed in liver tissue To explore the full spectrum of VEGFA isoforms expressed in HCC and NT tissue, we performed semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis using a preliminary panel of 20 HCC cases
We used VEGFA-iso primers that flank the variable region of VEGFA mRNA and amplify several PCR products of different lengths corresponding to certain VEGFA variants (Fig. 1A) The major isoforms expressed in all examined NT and most of HCC specimens were VEGFA-189 (523 bp), VEGFA-165 (451 bp), and VEGFA-121 (319 bp) (Fig. 1; Fig. S1)

Summary

Introduction

Hepatocellular carcinoma (HCC) is the most common and aggressive form of primary liver tumor and ranks second place in cancer-related mortality rates (Llovet et al, 2016). Angiogenesis is one of the most critical processes involved in HCC pathogenesis (Liu et al, 2017) It is mostly stimulated via the vascular endothelial growth factor (VEGF) signaling pathway activated by secreted VEGF proteins interacting with membrane tyrosine-kinase receptors KDR and FLT-1 (Ferrara, Gerber & LeCouter, 2003; Shen, Hsu & Cheng, 2010). Methods: A total of 50 pairs of HCC and non-tumor tissue samples were used to evaluate the VEGFA isoform spectrum using RT-PCR and quantitatively estimate changes in isoform expression using RT-qPCR. Correlations between these changes and tumor clinicopathological characteristics were analyzed. But not expression levels, of VEGFA-189 (decrease) and VEGFA-121 (increase) correlated with advanced Tumor-Node-Metastasis (TNM) and Barcelona Clinic Liver Cancer

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