Date of Award

12-4-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Bioinformatics

First Advisor

Larry Suva

Abstract

Bone metastasis is a common complication of breast cancer that significantly compromises patient survival due, in part, to the advanced stage of disease at time of detection. Early detection is key to patient survival, however, despite intense investigation in both the metastatic and early breast cancer setting there are currently no high performance biomarkers that can identify or predict the development of bone metastasis. In order to improve patient outcomes new diagnostic tools that can detect skeletal lesions in their earliest stages of development and/or identify patients at risk for developing bone metastases that may benefit from adjuvant anti-resorptive treatment for skeletal protection are desperately needed. To address this issue, first, a previously identified SELDI-based plasma protein signature indicative of breast cancer metastasis to bone was blindly and repeatedly validated in a third independent cohort of metastatic breast cancer patients (n = 34) with high sensitivity and specificity (Sn: 91%, Sp: 93%, AUC: 0.85). Importantly, the top discriminatory protein peak elevated in the plasma of bone metastasis patients was identified via PTHrP-specific immunodepletion as a specific and unique N-terminal 12 – 48 fragment of parathyroid hormone-related protein, namely PTHrP(12-48). This is the first identification of an in vivo circulating fragment of PTHrP in breast cancer patients and a major discovery for the bone metastasis field that will lead to new insight into PTHrP metabolism and the pathophysiology of breast cancer progression in bone. Given PTHrP established role in the vicious cycle of bone metastasis the translational utility of PTHrP(12-48) as a clinical marker and/or potential therapeutic target for bone metastasis in breast cancer was evaluated. Using a novel SELDI-based assay PTHrP(12-48) was identified to circulate at detectable levels in metastatic breast cancer patients, with significant elevations measured in breast cancer patients with bone metastases (102.5 ± 10.4ng/mL) compared to those without bony involvement (53.2 ± 5.2ng/mL). Evaluation of the diagnostic potential of PTHrP(12-48) via ROC curve analysis and logistic regression modeling identified PTHrP(12-48) to be a sensitive standalone marker for the presence of bone metastasis (Sn: 90%, Sp: 67%, AUC: 0.85) and to improve the specificity of bone metastasis detection by existing clinical measures, namely serum NTx (Sn: 86%, Sp: 95%, AUC: 0.99). PTHrP(12-48) bioactivity was investigated in silico using sequence- and structure-based Bioinformatics & Computational Biology techniques, and then tested in relation to bone cell differentiation in vitro. Cleavage site analysis identified lysine-specific monobasic and post prolyl endoproteases as likely candidates involved in PTHrP(12-48) processing, suggesting PTHrP(12-48) may be actively secreted by breast cancer cells. Structural modeling predicted PTHrP(12-48) to form an alpha helical core followed by an unstructured region after residue 40 or 42. Structure alignment and molecular docking simulations suggest PTHrP(12-48) is unlikely to have any productive biological interaction with the type I PTH receptor, and may target an alternate N-terminal PTHrP receptor. A nuclear localization signal (residues 19 – 21) and CDK2 phosphorylation site at Ser43, were also predicted suggesting PTHrP(12-48) could also function intracellularly or as an intracrine effector. Supporting the in silico predictions, PTHrP(12-48) had no stimulatory or inhibitory effect on osteoblast or adipocyte differentiation from PTH1R+ human mesenchymal stem cells, however, RANKL-induced osteoclastogenesis was significantly inhibited. This is the first documentation of an in vivo species of PTHrP having a direct effect on osteoclasts. Interestingly, immunostaining of classic N-terminal PTHrP target tissues (breast, placenta, cartilage) and paired primary human breast cancers and matched bone metastasis specimens using a PTHrP(12-48)-specific antibody suggests that PTHrP(12-48) not only circulates at detectable levels in metastatic breast cancer patients, but is commonly expressed by both primary breast tumors and bone metastases, but not normal breast tissue. Ultimately, these findings suggest that PTHrP (12-48) is present in blood plasma when the patient has breast cancer and that higher levels suggest that bone metastasis and/or metastatic progression may also be present. Further study must be performed to determine if, in the distant future, a simple blood test can be performed annually on women across the globe to detect PTHrP(12-48) and determine if they are either at risk for developing or currently have undiagnosed breast cancer or bone metastasis.

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