ORCID

http://orcid.org/0000-0002-4025-3091

Date of Award

Spring 5-15-2022

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Breast cancer is the most common non-cutaneous malignancy in women, with over 250,000 patients diagnosed each year in the United States alone. The bone is the most common site of recurrence in breast cancer, affecting over two-thirds of patients with metastatic disease and presenting as the only evidence of distant spread in up to 30%. Clinically, breast cancer bone metastases manifest most often as profoundly osteolytic lesions that negatively impact survival and patient quality of life. Bone-targeted therapies such as bisphosphonates and the anti-RANKL monoclonal antibody denosumab have revolutionized the treatment of bone metastases through inhibition of osteoclast-mediated bone destruction; however, these agents have failed to improve survival in the majority of patients. While other therapeutic options such as systemic chemotherapy exist, response to treatment is difficult to quantify, and bone metastases frequently exhibit resistance to anti-tumor interventions. Better, more specific therapies for these patients are urgently needed. Compared to common sites of visceral metastasis in breast cancer such as the lung and liver, the bone microenvironment represents a highly unique tumor niche, with distinct biophysical parameters, cellular and extracellular matrix (ECM) composition, and nutrient and chemical milieu. It is increasingly clear that interaction with this singular microenvironment promotes bone-specific tumor phenotypes that can be therapeutically exploited. In another study focused on delivering the common breast cancer chemotherapeutic docetaxel (DTX) to the bone metastatic microenvironment, we found that micelle nanoparticles targeted against the αvβ3 integrin heterodimer preferentially homed to bone metastases. To our surprise, this organ-specific targeting was driven not by microenvironmental αvβ3, but rather by upregulation of the integrin β3 (β3) subunit on bone-resident breast cancer cells not observed in visceral metastatic sites. Integrin heterodimers recognize ligand moieties present in the ECM and can initiate downstream signaling events with a wide array of consequences for cellular function. Tumoral αvβ3 integrin expression can promote bone metastasis and initiation of osteolysis, but its functional role in established metastases was largely unknown, particularly in the setting of chemotherapy. To investigate this aspect of bone metastatic biology, we generated Itgb3 knockout derivates of two bone-tropic murine breast cancer cell lines. While we found minimal differences in resistance to DTX in vitro, β3KO cells were significantly more sensitive to DTX attenuation in the bone microenvironment, and rescue of β3 expression in a β3KO clone restored resistance in the bone in a signaling-dependent manner. Ultrastructural, transcriptomic, and functional analyses revealed a β3-mediated alternative metabolic response to DTX characterized by increased protein production, oxygen consumption, and reactive oxygen species (ROS) generation. mTORC1 inhibitors, either free or loaded into αvβ3-targeted nanoparticles, could be combined with DTX to counteract this response and synergistically attenuate bone metastases. Our findings highlight the importance of the bone microenvironment as a driver of therapy resistance and provide proof of principle for a new, bone-specific combination therapy. We were also interested in elucidating the molecular mechanism responsible for upregulation of β3 on tumor cells in the bone microenvironment. A bone factor screen uncovered TGF-β as a candidate, and in vitro and in vivo inhibitor experiments confirmed the necessity of canonical TGF-β signaling through SMAD2/3 for tumoral β3 upregulation in bone metastases. Although TGF-β is known to be present at its highest concentration in the bone ECM, it is ubiquitous in most tumor microenvironments, prompting us to consider differences in active TGF-β bioavailability as the most important factor for β3 upregulation in metastatic cells. To explore this, we developed a TGF-β-responsive dual-luciferase reporter breast cancer cell line to use in the direct detection of TGF-β activity. Importantly, in a mouse model of Marfan syndrome with elevated active TGF-β in certain tissues, lung metastases exhibited elevated tumoral β3 expression and were resistant to DTX compared to similar tumors in wild type mice. These results establish bioavailable TGF-β as the causal microenvironmental factor in tumoral β3 upregulation and cement the importance of tumoral β3 for resistance to chemotherapy in breast cancer metastases. Together, our work demonstrates the profound influence of the microenvironment on tumor phenotype, even among different metastases present in the same animal. Future work focusing on direct targeting of both the β3-mediated alternative metabolic response to DTX and TGF-β signaling, as well as more basic questions surrounding the biology of tumor adaptations to treatment in the bone microenvironment, will be crucial for development of more effective therapeutic interventions for patients with bone metastases.

Language

English (en)

Chair and Committee

Katherine N. Weilbaecher

Committee Members

Abdel K. Azab

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