Basic and translational studies on species B adenoviruses

Human adenoviruses (Ads) have been classified into six species (A to F) currently containing 55 serotypes. For almost 2 decades vectors derived from group C serotype Ad5 have been extensively used for gene transfer studies. These Ad5 based vectors are able to efficiently infect many mammalian cell types (including both mitotic and post-mitotic cells) through interaction with a primary attachment receptor, the coxsackie and adenovirus receptor (CAR). Despite the many advantages of Ad5 based vectors a number of limitations have affected their therapeutic application to many diseases. Although they can transduce many tissue types, Ad5 based vectors are unable to efficiently transduce several potential disease target cell types, including hematopoietic stem cells and malignant tumor cells. Therefore, newer vectors have been developed based on Ad serotypes other than Ad5. This thesis focuses on species B Ads. Species B Ads are comprised of three groups based on their receptor usage. Group 1 of species B Ads (Ad16, 21, 35, 50) nearly exclusively utilize CD46 as a receptor; Group 2 (Ad3, Ad7, 14) share a common, unidentified receptor/s, which is not CD46 and which was tentatively named receptor X; Group 3 (Ad11) preferentially interacts with CD46, but also utilizes receptor X if CD46 is blocked. Species B group Ads are important human pathogens. Species B group 2 serotypes are isolated from patients with respiratory tract infections, whereas the Group 1 viruses are described as causing kidney and urinary tract infections. B-group Ad infections often occur in immunocompromised patients, including AIDS patients, recipients of bone marrow transplants, or chemotherapy patients. Recent studies performed in U.S. military training facilities indicate an emergence of diverse species B serotypes at the majority of sites. This included the group 1 serotype 21 and the group 2 serotypes 3, 7, and 14. CD46-targeting vectors derived from Ad35 and Ad11 are important tools for in vitro gene transfer into human stem cells, including hematopoietic stem cells and induced pluripotent stem cells. Ad35 and Ad11 have been used as tools for cancer therapy, because CD46 appears to be uniformely overexpressed on many cancers. Furthermore, receptor X-targeting vectors, i.e vectors derived from Ad3 or vectors containing Ad3 fibers have shown superior in the transduction of tumor cells both in vitro and in vivo and are currently being used clinically in cancer patients. While extensive basic virology studies have been done on Ad5, the information of species B group 1 interaction with CD46 is limited. Furthermore, the receptor for a major subgroup of species B Ads (receptor X) is unknown. The goal of this thesis was it therefore to better understand virological and translational aspects of species B Ads. The specific findings described in this thesis include i) the identification of CD46 binding sites within the Ad35 fiber knob, ii) the study of the in vitro and in vivo properties of Ad vectors with increased affinity to CD46. iii) the study of the receptor usage of a newly emergent Ad14a, iv) the identification of desmoglein 2 as the receptor for Ad3, Ad7, Ad11, and Ad14, v) the delineation of structural details of Ad3 virus interaction with DSG2, and vi) the analysis of functional consequences of Ad3-DSG2 interaction. As a result of these basic virology studies two Ad-derived recombinant proteins have been generated that can be used to enhance cancer therapy by monoclonal antibodies.

Role of CIP2A in carcinogenesis

Worldwide and notably in the developed countries, cancer is an increasing cause of morbidity and mortality, being the second most common cause of death after ischemic heart disease. Now and in the future new cancer cases need to be diagnosed earlier. Prognostic factors may be helpful in recognizing and handling those patients who need more aggressive therapy, and it is also desirable to predict treatment response accurately. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein predominantly expressed in malignant tissues and inhibiting protein phosphatase 2A (PP2A) activity; it is a promising target for cancer therapy. The aim of this thesis was to evaluate the prognostic role of CIP2A in solid cancers, and for this purpose to explore expression of CIP2A, and investigating regulation of CIP2A in order to gain insight into signalling pathways leading to alteration in prognosis. Patients diagnosed with gastric, serous ovarian, tongue, or colorectal cancer at Helsinki University Central Hospital were included. Tumour tissue microarrays assembled from specimens from these patients were prepared and stained immunohistochemically for CIP2A protein expression. Associations with clinicopathologic parameters and other biomarkers were explored, and survival analyses were done according to the Kaplan-Meier method. Study of the role of CIP2A in intracellular signalling in vitro involved gastric, ovarian, and tongue cancer cell lines. We found CIP2A to be highly expressed in gastric, ovarian, tongue, and colorectal cancer specimens. CIP2A was associated with clinicopathologic parameters characterizing an aggressive disease, namely advanced stage, high grade, p53 immunopositivity, and high proliferation index. CIP2A led to recognition of gastric, ovarian, and tongue cancer patients with poor prognosis, however, with a cancer type-specific cut-off level for prognostic significance. In tongue cancer, it served as an independent prognostic marker. In contrast, in colorectal cancer, CIP2A provided no prognostic value. In cancer cell lines, CIP2A was highly expressed at both protein and mRNA levels, and promoted cell proliferation and anchorage-independent growth. In gastric cancer, we demonstrated with a MYCER construct in mouse embryo fibroblasts that activation of MYC led to increased CIP2A mRNA expression, and hence we suggested that a positive feedback mechanism between CIP2A and MYC may potentiate and prolong the oncogenic activity of these proteins. We demonstrated in ovarian cancer an association between CIP2A and EGFR protein overexpression and EGFR gene amplification. In ovarian and tongue cancer cells we showed that depletion of EGFR downregulates CIP2A expression. In conclusion, high CIP2A expression occurred frequently among patients with aggressive disease. CIP2A may serve as a prognostic marker in gastric, ovarian, and tongue cancer and thus may help in tailoring therapy for cancer patients. The positive feedback mechanism between CIP2A and MYC, as well as the positive regulation of CIP2A by EGFR, are a few signalling pathways regulating and regulated by CIP2A. These and other mechanisms need to be studied further, however. CIP2A is a potential target for therapy, and its potential role as predictive marker and as a tumour marker in serum requires exploration.