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LAURA SAVARIAU - Final Dissertation Defense - HUGEN PhD Candidate

Department of Human Genetics Doctoral Candidate, Laura Savariau, B.S., defends her dissertation on “E-Ccadherin Loss and Other Drivers of Invasive Lobular Carcinoma” 

ADVISOR: STEFFI OESTERREICH, PHD

Committee Members:

  • Zsolt Urban, PHD
  • Beth L. Roman, PHD
  • Brenda Diergaarde, PHD

 

ABSTRACT:

Invasive lobular carcinoma (ILC) is the second most frequently diagnosed histologic subtype of invasive breast cancer following no special type carcinoma (NST) and accounts for 10-15% of all cases. The pathognomonic hallmark of ILC is the genetic loss of E-cadherin (CDH1) causing the disruption of adherens junctions and resulting in discohesive, linear growth of ILC cells. Beyond the loss of E-cadherin effect on this unique histology, there is limited data on its role in ILC metastasis. Therefore, we generated three ILC cell lines; MDA-MB-134-VI (MDA-MB-134), SUM44PE (SUM44), and BCK4 with inducible E-cadherin overexpression that successfully restored adherens junctions. E-cadherin expression reduced growth in 2D culture which was even more evident in 3D ultra-low attachment conditions where increased cell death was also observed, consistent with the previously described role of E-cadherin in anoikis. E-cadherin expression failed to increase the poor migratory and invasive ability of ILC cell lines as measured by transwell assays. However, E-cadherin expression in SUM44 led to decreased haptotaxis to collagen I, while increasing its adherence to collagen I. Clinically, ILC tumors have increased propensity to metastasize to the gastrointestinal tract, brain and ovaries compared to NST tumors. Although E-cadherin expression did not significantly affect initial tumor growth following orthotopic injection into the mammary fat pad of NSG mice, E-cadherin expression reduced metastatic seeding in MDA-MB-134 and altered metastatic organotropism in both MDA-MB-134 and SUM44.

To further understand metastases in patients with ILC, I studied ILC cell line xenograft murine models, and followed-up on our recent molecular analysis of clinical ovarian metastases from breast cancer patients. Our group recently developed human ILC cell line xenograft murine models which developed spontaneous metastases to brain, bone and other sites including those frequently seen in patients with ILC such as the ovary. To explore organotropism in these models, I performed transcriptomic analysis of the xenograft model primary tumors, brain and ovarian metastasis and identified enrichment of genes involved in extracellular matrix remodeling in the primary tumor, and increased expression of the known brain metastasis driver RET in the brain metastases. Inclusion of normal mammary gland, brain and ovary tissue in the transcriptomic analysis enabled identification of up-regulation of prolactin and growth hormone in the murine tumor microenvironment.

Our transcriptomics studies of ovarian metastases which are enriched in patients with ILC revealed increased Calcium Sensing Receptor (CaSR) expression in ovarian metastasis. To understand the possible tumorigenic phenotypes conferred by CaSR, I used CaSR over-expression ILC cell lines and observed that while the CaSR alone does not increase growth or support cell migration, upon activation with calcium or calcimimetic (R568) CaSR promoted migration which was abrogated by treatment with a calcilytic (NPS2143). Immunofluorescence microscopy revealed increased F-actin staining following CaSR activation and was reversible with calcilytic treatment.  Activated CaSR triggered the MEK/ERK signaling pathway, and its inhibition abrogated the migratory phenotype and F-actin increase. Lack of migration with hormone deprivation showed that the activated CaSR requires estrogen receptor activation for migration.

Collectively, my work as part of this thesis created and characterized novel cell line and xenograft models to improve our understanding of the hallmark loss of E-cadherin and other drivers of ILC, which I hope will ultimately enable the development of more effective therapies and improve the outcome of patients with this understudied histological subtype of breast cancer.

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