Ozyamaci, Ozlem (2023) The biological significance, targeting and prognostic significance of the HER family members in liver cancer. (PhD thesis), Kingston University, .
Abstract
Liver cancer is renowned for its high mortality rates. Worldwide, it is the third leading cause of cancer-related deaths. Although there has been a considerable improvement in the range of treatments available, there is still no cure or long-term treatment plan for advanced HCC, with the tumour heterogeneity being a major cause of treatment failure. Therefore, further research is essential to gain a better understanding of the complex biology of liver cancer, to overcome some of the limitations of the current treatment options and to develop novel and more effective therapies against HCC. In the past few decades, increased expression, and activation of the human epidermal growth factor receptor (HER) family members have been detected in various cancer types. Of these, EGFR and HER-2 are two important therapeutic targets for the treatment of cancer patients with the small molecules tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs) based drugs. However, there is currently no comprehensive study of all members of the HER family, their targeting and clinical significance in liver cancer, which is a major aim of this PhD project. In this study, the effect of several TKIs targeting one or more members of the HER family and other growth factor receptors and Cyclin dependent kinases (CDKs) were investigated for their effects on the growth and migration of a panel of human liver cancer cell lines (LCCLs) in comparison to the effect of two of the FDA approved drugs sorafenib, regorafenib, and chemotherapy, doxorubicin. The possible association between the expression levels of the HER family members and the putative liver cancer cell stem biomarker CD44 and the response to the treatment with such agents were also investigated. At the maximum concentration of 10uM used in this study, of all the HER family TKIs, treatment with the irreversible EGFR/HER-2/HER-4 TKI afatinib was able to inhibit the proliferation of two of the seven human LCCLs. Treatment with afatinib has also inhibited the migration of the liver cancer cells. The other HER TKIs tested had limited inhibitory response and again were incapable on inhibiting the proliferation of all LCCLs. Of the other targeted agents, the CDK 1/2/5/9 inhibitor dinaciclib, had the greatest inhibitory effect on proliferation and migration of the liver cancer cells. The expression level of all members of the HER family and CD44 were determined by flow cytometry. While the expression of the HER family members was low in the human LCCLs, four of the seven LCCLs had overexpression of CSC marker CD44 and the majority LCCLs were found to be IGF-IR and C-Met positive. With the exception of the association between HER-3 and response to afatinib, no clear association was found between the expression of HER family members and the response to the treatment with the other types of the HER inhibitors in the LCCLs. Interestingly, while treatment with a combination of afatinib and sorafenib resulted in the synergistic growth inhibition of one LCCL, the same combination was found to be antagonistic in another cell line, highlighting the complex biology of liver cancer. Finally, the expression pattern and prognostic significance of all members of the HER family, CD44, EpCAM and the tumour cell proliferation biomarker Ki67 were investigated in the tumour specimens from 43 patients with hepatocellular carcinoma. Of the 43 patients, tumours from 35%, 58%, 19%, and 26% of the cases were EGFR, HER2, HER4 and EGFRvIII positive respectively. Interestingly, all tumours were found to be HER-3, IGF-IR or c-MET negative whereas 23% of the cases had co-expression of both EGFR and HER-2. Of the putative cancer stem cell biomarkers, 40% and 33% of the cases were CD44 and EpCAM positive. Of these, the expression of EGFRvIII and membranous expression of CD44 were associated with a poorer survival in patients with hepatocellular carcinoma. In contrast, the expression of cytoplasmic CD44 and HER-2 immunostaining of 1+ intensity was associated with a better overall survival. Taken together, the results of this investigation suggest that the heterogeneous expression of these biomarkers attributes to the response of liver cancer cells to the treatment with HER inhibitors and further investigations are warranted on the prognostic significance of EGFRvIII and CD44 and the therapeutic potential of the HER inhibitors when used in combination with other targeted therapies in the treatment of the HER family, EGFRvIII and CD44 positive liver cancer.
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