The lack of effective medical treatment for the adrenocortical carcinoma (ACC), induce to search a better medical treatment protocols for ACC neoplasm. Based on the efficacy of sorafenib, a tyrosin kinase inhibitor, in different human tumors, the aim of this study was to understand the mechanism through which sorafenib acts on ACC and sometimes it alone is not able to induce a long-lasting antiproliferative effect in this tumor.
The effects of sorafenib will be tested on ACC primary cell culture from adrenalectomy and parallely in H295R cell line by evaluating cell viability and apoptosis and the VEGF receptor signaling, such as VE-cadherin and ß-catenin complex formation. In addition we will test the sorefenib effects on a 3D cell culture model by using the same H295R model in vitro. Cell death will be investigated and experiments of co-immunoprecipitation will be performed in order to clarify the involvement of VEGFR-VE-cadherin complex and b-catenin proteins after sorafenib treatment. The results obtained will be confirmed both by the ultrastructural analysis and by 3D model in ACC cells.
Sorafenib is an inhibitor of several receptor tyrosine kinases involved in neovascularization, including VEGFR2, VEGFR3, and platelet-derived growth factor (Hasskarl J, Cancer Res '10), it has shown efficacy against a wide variety of tumours (Llovet JM et al, N Engl J Med '08; Keating GM et al, Drugs. '09). Since sorafenib showed a broad spectrum of antitumor activity in the preclinical studies (Cheng AL et al, Lancet Oncol '09; Bernini GP et al, JCEM '02; Mariniello B et al, ERC '12), multiple clinical trials are undertaken to further investigate the role of this drug alone or in combination with chemotherapy for the treatment of several neoplasm (Wilhelm SM et al, Cancer Res '04;Cheng AL et al, Lancet Oncol '09). For these characteristics sorafenib has been approved by the FDA in order for the treatment of patients with advanced renal cell carcinoma, with unresectable hepatocellular carcinoma and for the treatment of locally recurrent or metastatic, progressive differentiated thyroid carcinoma refractory to radioactive iodine treatment.
Despite the antineoplastic effects described for sorafenib, some patients may exhibit neoplastic progression during the therapy with this drug as demonstrated by the comparison of the progression-free survival curves between treated patients and controls. Finally numerous animal studies have also suggested that antiangiogenesis drugs may, in certain situations, actually accelerate metastatic spread which is recognized as a new form of adaptive resistance of cancer cell (Ebos JM et al, Cancer Cell '09).
ACC is a highly vascularized neoplasia (Else T et al, Endocr Rev '14) and metronomic chemotherapy is thought to exert its anticancer activity mainly by inhibiting tumor angiogenesis. Moreover, the antiangiogenic activity of metronomic chemotherapy can be theoretically increased by the concomitant administration of an antiangiogenetic drug (Loges S et al, Cancer Cell '09). Therefore, there is a strong rationale for testing the combination of metronomic chemotherapy plus antiangiogenetic drugs in the management of ACC patients.
Berruti et al. tested the combination of daily sorafenib and weekly paclitaxel therapy in patients affected by metastatic ACC in progression after treatment with mitotane in combination with one or two chemotherapy lines (Berruti A et al, Eur J Endocrinol '12) in a multicenter, prospective phase II trial. The result of this experience documented a clear progressive disease in nine consecutive patients at the first restaging dose after 2 months, and this led to interruption of the clinical trial. These data suggest that this combination therapy may have paradoxically favored the tumor progression. Although the data regarding the progression of tumors during treatment with sorafenib are present in the literature (Zhang W et al, Gastroenterology '12) few data are instead available with regard to mechanism by which sorafenib can elicit a malignant phenotype. At this regard he primary objective of our study will be to determine in vitro model whether sorafenib was able to induce a malignant phenotype in ACC.
For this purpose we will conduct the experiments both in primary ACC cell culture and in 2-3D models. We chose the in vitro 3D cultures, since it represent an useful approach for studying the invasive proprieties and metastatic potential of tumor cells, thus facilitating the development and screening new drugs (Zietarska M et al, Molecular Carcinogenesis '07).
We will study if sorafenib induces a cell growth inhibition characterized by induction of a significant cell death with particular reference to the intercellular junctions connected to VE-cadherin and B-catenin proteins.
In addition we will investigate the molecular processes involving the angiogenetic factor associated to tumor progression.
Cell culture model to grow tumour spheroids in suspension will be used to study the responses of ACC to sorafenib treatment. H295R cells have the capacity to form spheroids, as reported by Lichtenauer UD and collegues (Horm Metab Res '13). Ability to form spheroid colony is a recognized method to identify cancer stem cells (CSC) displaying an enhanced tumorigenic ability (Kasper S et al, Urol Oncol '09). This specific cell population is thought to be closely linked to the epithelium-mesenchymal process (EMT) defined as crucial in metastatic spread and in tumor recurrence as well. In cancer, EMT is associated with poor survival for the patients and seems to be a key step in the development of metastasis, since cells lose their polarity and cell-to-cell contacts and therefore become more motile (Mani SA et al, Cell '08).