Role of neutrophils in breast cancer metastasis
Creators: name: Kulkarni, Aneesha
Publisher: name: Purdue University Graduate School
Year: 2023
Subjects: classificationCode: 321101
subject: Cancer cell biology
classificationCode: 321111
subject: Solid tumours
classificationCode: 320409
subject: Tumour immunology

description: Breast cancer remains a major cause of cancer-related deaths among women despite several advances in the field due to metastasis with a 5-year survival rate of less than 30% for metastatic breast cancer. Dissemination of tumor cells to metastatic sites begins as early as the primary tumor is diagnosed at just 4mm in size. These cells remain dormant for extended periods of time evading immune surveillance and later turn into therapy resistant metastases resulting in the poor prognosis in breast cancer patients. Hence, there is a <b>critical need </b>to improve our understanding of the metastatic programs in breast cancer and its contributors to develop better therapy options.One such contributor is alcohol which is listed as a carcinogen by the National Toxicology Program. Alcohol consumption is a risk factor for several cancers and increases the risk of breast cancer incidence in a dose dependent manner. We have observed in preliminary studies, that alcohol consumption causes increased neutrophil extracellular trap (NET) formation in the lungs and outgrowth of previously dormant cancer cells in mice. Further, NETs increase cancer cell seeding and play a role in metastasis. Hence, we hypothesized that alcohol consumption breaks cancer cell dormancy by activating neutrophils.In this study, we have broken cancer cell dormancy and generated a novel cell line, Alcohol-D2.OR, by inducing outgrowth of the dormant D2.OR cells in mice through alcohol consumption. Reinjection of the Alcohol-D2.OR cells, into alcohol-naïve mice results in aggressive outgrowth of the cells suggesting these cells are modified on a genetic level. Indeed, RNA sequencing analysis of the gene expression in the cells showed that these cells have significantly modified gene expression as well as modified morphology and surface protein expression than the parental D2.OR cells. Importantly, from our analysis we have identified a tumor suppressor, SPINK5 which was significantly downregulated in the alcohol line. Further, SPINK5 expression in cancer cells suppressed neutrophil activity in-vitro. Knockdown of SPINK5 in the parental D2.OR line resulted in outgrowth of the cells in-vivo with increased lung NETs highlighting the importance of this gene for maintenance of dormancy by suppression of neutrophil activity.Hence, we have successfully identified a gene responsible for dormancy maintenance, SPINK5 which will aid in not only therapeutic intervention but also in identification of breast cancer patients likely to progress to metastasis. Further, the newly established Alcohol-D2.OR cells provide a novel tool to study other initiators of metastasis in breast cancer.A common side-effect of most chemotherapeutic treatments is neutropenia, reduced neutrophils in circulation increasing susceptibility to infections. Hence, GM-CSF is often administered to patients to mobilize bone marrow neutrophils. However, neutrophils have been increasingly shown to promote distant metastases. Circulating disseminated cancer cells (DCCs), which are present as early as primary diagnosis, have been shown to activate neutrophils resulting in the release of neutrophil extracellular traps (NETs). These NETs alter the lung architecture providing a suitable environment for the seeding and growth of DCCs promoting lung metastases. One key player in neutrophil activation is spleen tyrosine kinase (SYK), an intracellular non-receptor kinase which is activated by the engagement of b-integrin on the neutrophil surface.Using a chemical genetics approach we are able to specifically inhibit SYK in the murine host. Using our transgenic model of specific SYK inhibition as well as the FDA approved SYK inhibitor, fostamatinib, we see similar results of decreased lung metastases compared to controls. We also observed decreased neutrophil viability in-vitro in the presence of fibronectin, an effect that was not seen on plastic highlighting the importance of integrin mediated activity of SYK. We also observe decreased neutrophil and macrophage infiltration into the lungs upon host-specific SYK inhibition. Overall, these findings suggest a paracrine effect of SYK in stromal cells that promotes favorable tumor microenvironment (TME) and its inhibition may be a useful therapeutic option to combat DCCs from forming metastases.Hence, through this work we address two mechanisms of neutrophil-mediated breast cancer metastasis and that therapeutic intervention by rescuing SPINK5 expression in cancer cells or inhibition of SYK in the tumor microenvironment can suppress pulmonary metastasis in breast cancer.