The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase

Tumour metastasis is a complex process involving reciprocal interplay between cancer cells and host stroma at both primary and secondary sites, and is strongly influenced by microenvironmental factors such as hypoxia. Tumour-secreted proteins play a crucial role in these interactions and present strategic therapeutic potential. Metastasis of breast cancer to the bone affects approximately 85% of patients with advanced disease and renders them largely untreatable. Specifically, osteolytic bone lesions, where bone is destroyed, lead to debilitating skeletal complications and increased patient morbidity and mortality. The molecular interactions governing the early events of osteolytic lesion formation are currently unclear. Here we show hypoxia to be specifically associated with bone relapse in patients with oestrogen-receptor negative breast cancer. Global quantitative analysis of the hypoxic secretome identified lysyl oxidase (LOX) as significantly associated with bone-tropism and relapse. High expression of LOX in primary breast tumours or systemic delivery of LOX leads to osteolytic lesion formation whereas silencing or inhibition of LOX activity abrogates tumour-driven osteolytic lesion formation. We identify LOX as a novel regulator of NFATc1-driven osteoclastogenesis,independent of RANK ligand, which disrupts normal bone homeostasisleading to the formation of focal pre-metastatic lesions. We show that these lesions subsequently provide a platform for circulating tumour cells to colonize and form bone metastases. Our study identifies a novel mechanism of regulation of bone homeostasis and metastasis, opening up opportunities for novel therapeutic intervention with important clinical implications

The Involvement of P2X7R Signalling in LOX Mediated Formation of a Pre-Metastatic Niche in Bone

Metastasis to bone seriously complicates treatment of cancer, resulting in an incurable and debilitating condition which can greatly decrease quality of life as a result of excessive bone destruction. Recent research has identified tumour secreted LOX as a contributor to cancer induced bone loss, by generating osteolytic lesions prior to cancer cell arrival, thus contributing to pre-metastatic niche formation, and enhancing metastatic cell recruitment and survival in bone. Previous research has also observed that LOX accumulates at areas of pressure, and is hypoxically regulated, highlighting a potential involvement with P2X7R, which contributes significantly to cancer cell survival and osteoclastic resorption of bone. This thesis builds on these observations by investigating the involvement of LOX and P2X7R in pre-metastatic modification of bone in a syngeneic BALB/c mouse/4T1 murine breast cancer model. Analysis of µCT data from the tibia of tumour bearing mice revealed significantly reduced bone destruction upon knockdown of LOX in the tumour, or knockout of P2X7R in the mouse, confirming an interaction. These observations were repeated in cancer cell free mice, receiving injections of conditioned medium from 4T1 cells grown in vitro, confirming that bone destruction and osteolytic lesion formation are the result of secreted factors from the primary tumour, and can occur prior to cancer cell arrival at the bone. The effect of mechanical loading upon LOX/P2X7R modification of pre-metastatic bone was also investigated by subjecting tumour bearing mice to non-invasive axial loading of the right hindlimb. Mechanical loading was found to have a mixed effect on bone, driving thickening of cortical and trabecular bone, but also osteolytic lesion formation. P2X7R contributed to bone response to loading, while tumour-secreted LOX had no effect. Taken together, mechanical loading and P2X7R inhibition were found to effectively combat tumour induced bone loss, identifying novel targets for future treatments of metastatic bone disease