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Characterizing the Biological and Functional Consequences of WWOX Dysregulation in Canine Osteosarcoma

Breitbach, Justin T
http://rave.ohiolink.edu/etdc/view?acc_num=osu159810524642971

Abstract Details

2020, Doctor of Philosophy, Ohio State University, Comparative and Veterinary Medicine.
Osteosarcoma (OS) is the most common primary bone tumor affecting children and dogs. While few patients have radiographic evidence of distant metastases at the time of diagnosis, it is well established that lung micrometastases develop early in the disease course. Despite aggressive surgery and chemotherapy, 30-40% of humans and 90% of dogs succumb to chemotherapeutic resistant metastatic disease. Both clinical and molecular evidence suggests that human and canine OS share several key features including tumor location, the development of chemotherapy resistant metastases, p53 mutations, and similar transcriptional profiles among others. Given the overlap with the complex biology of human OS, canine OS represents a relative model to investigate the biology of the disease. The purpose of this work was to evaluate the influence of dysregulation of the tumor suppressor gene, WW domain-containing oxidoreductase (WWOX), on osteosarcoma biology as it is commonly lost or attenuated in the human disease. The first objective was to determine whether WWOX expression was reduced in canine OS and whether loss of WWOX could confer phenotypic effects in OS cells. We identified that WWOX is reduced in 57% of canine OS tumors, findings of which are concordant to the human disease. In addition, we demonstrated that loss of WWOX can influence the phenotype of OS, specifically by enhancing invasion and proliferation. Furthermore, we determined that double-stranded DNA damage influences WWOX expression and in the absence of WWOX, OS cells are more resistant to DNA damaging agents. These data suggest that loss of WWOX expression is a common event in canine OS which may contribute to the chemoresistance commonly seen in the disease. Our second objective was to evaluate the effects of osteoblast specific ablation of WWOX in a novel mouse model as previous models have revealed increased OS formation and bone defects. To promote osteoblast-specific deletion of WWOX, transgenic WWOX-floxed mice were crossed with Col1a1-Cre transgenic mice which express the Cre-recombinase gene under control of the Col1a1-promoter whose expression is restricted to early committed osteoblasts. Via qRT-PCR and Western blot analysis, we demonstrated osteoblast specific deletion of WWOX from primary osteoblast cultures generated from the calvaria of knock-out mice. Additionally, evaluation of osteoblasts deficient in WWOX revealed enhanced proliferation compared to wild type osteoblasts. Though no obvious phenotypic differences in bone formation were identified between wildtype and knockout mice via faxitron analysis, this mouse model is a useful tool for future studies evaluating the effects of WWOX on osteoblast biology and bone formation. Lastly, our third objective was to evaluate the in-vitro effects of the novel SINE compound, veridnexor, in canine OS cells. We show that the major nuclear export protein, exportin 1 or XPO1, is overexpressed in canine OS tumors and cell lines, which indicated to us that it was a possible relevant target for therapeutic intervention. We subsequently treated OS cells with increasing doses of the XPO1 inhibitor, verdinexor, and determined IC50 values to be in low nanomolar concentrations. Additionally, verdinexor induced apoptosis in canine OS cells and resulted in downregulation of XPO1 protein, suggestive of direct inhibition of XPO1. Furthermore, we determined that verdinexor works synergistically with the standard of care chemotherapeutic agent doxorubicin to induce cell death, possibly though inhibition of DNA damage repair proteins. Collectively, these data suggest that use of verdinexor and doxorubicin together may be a relevant treatment strategy in canine patients with OS. These studies provide evidence supporting the notion that decreased WWOX expression is a common event in canine OS which may possibly contribute to chemotherapy resistance seen in this disease. Additionally, future concurrent use of the SINE compound verdinexor with doxorubicin may prove to be an important therapy for dogs with chemotherapeutic resistant disease.
Joelle Fenger, DVM, PhD (Advisor)
Stefan Niewiesk, DVM, PhD (Committee Member)
Maxey Wellman, DVM, PhD (Committee Member)
Sue Knoblaugh, DVM (Committee Member)
Ryan Roberts, MD, PhD (Committee Member)
138 p.
Cellular Biology; Molecular Biology; Oncology
canine osteosarcoma; cancer; WWOX; verdinexor; XPO1

Recommended Citations

Citations

  • Breitbach, J. T. (2020). Characterizing the Biological and Functional Consequences of WWOX Dysregulation in Canine Osteosarcoma [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu159810524642971

    APA Style (7th edition)

  • Breitbach, Justin. Characterizing the Biological and Functional Consequences of WWOX Dysregulation in Canine Osteosarcoma. 2020. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu159810524642971.

    MLA Style (8th edition)

  • Breitbach, Justin. "Characterizing the Biological and Functional Consequences of WWOX Dysregulation in Canine Osteosarcoma." Doctoral dissertation, Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu159810524642971

    Chicago Manual of Style (17th edition)

osu159810524642971
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