title: The Role of Post-Menopausal Osteoporosis in Bone Metastatic Breast Cancer and the Effect of Monocyte Chemoattractant Protein-1
Creators: name: Mulholland, Bridie S
Publisher: name: Griffith University
Year: 2021
Subjects: subject: Interrelatedness
subject: Interrelation
subject: Osteoporosis
subject: Metastatic breast cancer
subject: monocyte chemoattractant protein-1
subject: MCP-1
subject: Chemokine

description: Osteoporosis is a debilitating disorder of the bone that is characterised by excessive bone resorption. Breast cancer is the most common cancer diagnosed in women globally and frequently metastasises to the bone. Skeletal lesions that form secondary to breast cancer are predominantly osteolytic which, like osteoporosis, are characterised by excessive bone resorption. Osteoporosis is most notably associated with the aging population and most prominently affects aging, post-menopausal women. Similarly, breast cancer is most common in women over the age of 50. With credit to many scientific discoveries, breast cancer has a high five-year survival rate. Unfortunately, the same cannot be said for metastatic breast cancer. Skeletal lesions that form secondary to breast cancer are exceptionally difficult to treat and manage, and are often associated with debilitating pain. Osteoporosis and osteolytic skeletal lesions are alike in their excessive preference of bone resorption, and anti-resorptive therapies that are typically used in the management of osteoporosis are often used in the management of skeletal lesions. The similarities between osteoporotic and osteolytic environments imparts a possibility that osteoporosis, as an underlying bone condition, may provide a favourable environment for the progression of breast cancer in bone. This thesis sought to explore the interrelatedness of osteoporosis and metastatic breast cancer in the context of monocyte chemoattractant protein-1 (MCP-1), a chemokine that has been implicated in both bone resorption and breast cancer progression. Given the similarities in age between post-menopausal osteoporotic women and women with breast cancer, and the use of the ovariectomy-induced osteoporosis model in Chapter 4 and Chapter 5, this thesis discusses osteoporosis in the context of post-menopausal osteoporosis. Chapter 3 explored the differences in the genetic profile of the 4T1.2 murine breast cancer cell line and its parent, the 4T1 murine breast cancer cell line. The 4T1.2 line is a clone of the 4T1 line that was selected and expanded based on its increased capacity for bone metastasis, and was used in Chapter 4 and Chapter 5 as a model of breast cancer bone metastasis. This chapter sought to characterise the genetic profile of the 4T1.2 line relative to the 4T1 line to identify differentially expressed genes that may contribute to the increased propensity of the 4T1.2 line to colonise in bone. In light of the classical meaning of the word clone, we showed that a number of genes were upregulated and downregulated in the 4T1.2 line relative to the 4T1 line using NanoString technology. Of the most noteworthy in the context of breast cancer bone metastasis, expression of BST2, CXCL10 and TGFB2 were upregulated in the 4T1.2 line. These genes have varying but important roles in breast cancer aggression, breast cancer bone metastasis and osteoclastogenesis. Coupled with the known high expression of PTHrP in the 4T1.2 line, the upregulation of these genes likely contributes to the 4T1.2 line’s preference for bone as a metastatic site. An additional aim of this chapter was to assess the appropriateness of using the 4T1.2 line as a model of breast cancer bone metastasis in Chapter 4 and Chapter 5. Chapter 5 introduced a dominant negative mutant of MCP-1 (7ND), to test if MCP-1 inhibition would reduce tumour burden in bone. As we wanted to introduce 7ND in the same manner that an anti-resorptive would be used to manage bone metastasis, we needed to ensure that the introduction of 7ND would not interfere with the metastatic potential of the 4T1.2 line by confirming that MCP1 and its receptor, CCR2, were not dominantly expressed. We showed that MCP1 expression was low in the 4T1.2 line and that CCR2 was not expressed at all. As such, we concluded that 7ND was unlikely to effect the metastatic potential of the 4T1.2 line and we were able to proceed with the animal model used in Chapter 4 and Chapter 5. Chapter 4 and Chapter 5 both utilised an ovariectomy-induced model of osteoporosis and the 4T1.2 model of breast cancer bone metastasis. Chapter 4 explored whether tumour burden in bone would be greater in ovariectomised mice than in sham-operated mice, given the mimicry of the osteoporotic and osteolytic bone environments. Chapter 5 explored whether the introduction of 7ND to inhibit MCP-1, as a resident component of the bone environment, would decrease the burden of tumour in ovariectomised and sham-operated bone. There were a number of limitations associated with this animal model that hindered our ability to draw conclusions in Chapter 4 and Chapter 5 and we were forced to accept the null hypothesis in both chapters. We highlighted these limitations as fundamental limitations associated with performing ovariectomy-induced osteoporosis models in aged mice that had reached peak bone mass and discussed the various implications of the methodological challenges. Chapter 6 explored whether MCP-1 expression in the primary breast tumour of women diagnosed with triple negative invasive ductal carcinoma was associated with incidence of distant relapse – metastasis – or breast cancer related-death with distant relapse. MCP-1 is widely regarded as a pro-metastatic chemokine and, as such, we hypothesised that high MCP-1 expression in the primary tumour would be associated with an increased risk of distant relapse and breast cancer-related death with distant relapse. We showed with initial Kaplan-Meier analysis that, in fact, low expression of MCP-1 in the primary tumour was associated with an increased risk of distant relapse and breast cancer-related death with distant relapse. Again, we were forced to accept the null hypothesis; however, this finding contradicted the overwhelming literature in support of MCP-1 as a pro-metastatic chemokine in breast cancer, and so we reconsidered our findings in the context of breast cancer stages. Interestingly, we noted that most low MCP-1 expressing primary tumours were Stage III or Stage IV cancers, and we highlighted a shift in MCP-1 expression from high expression in Stage I triple negative breast cancers to low expression in Stage IV triple negative breast cancers. We noted that it is critical this shift in expression is characterised, as there is the possibility the shift may be mechanistic and drive, in part, the metastasis of triple negative breast cancers, potentially rendering anti-MCP-1 anti-metastatic therapies unsafe in some patients with triple negative breast cancer. Overall, this doctoral thesis has highlighted a compelling need for improved characterisation of the role MCP-1 plays in metastatic breast cancer progression and how this role may implicate other conditions, such as osteoporosis, in breast cancer bone metastasis. Targeting MCP-1 has promise as a therapeutic, particularly in the emerging field of targeted chemokine therapies. The lack of studies that assess and define the clinical risk that co-morbidities of bone may have on breast cancer bone metastasis is alarming, especially in the case of osteoporosis, a preventable disease that is regularly underdiagnosed.