Monday, March 31, 2025
3/31/2025
Modeling Metastasis and Acquired Drug Resistance Using Circulating Tumor Cells - PROJECT SUMMARY Advanced hormone receptor positive (HR+) breast cancers, the most common subtype, are initially responsive to multiple endocrine interventions, but they ultimately develop drug resistance. Circulating tumor cells (CTCs) underlie the blood-borne metastatic spread of cancer, and they also provide a noninvasive source to sample tumor cells during the course of therapy and acquired resistance. Using a microfluidic enrichment technology that preserves CTC viability, we successfully established a cohort of 15 CTC-derived longterm cultures from women with refractory HR+ breast cancer. A subset of these shows the expected acquired ESR1 and PIK3CA mutations, confirmed in matched metastatic tumor biopsies. However, the most common, and least well understood, correlate of endocrine resistance is loss of estrogen receptor-α (ER) expression by cancer cells. In these cases, cultured CTCs recapitulate ER silencing, and some are biphenotypic, with coexisting ER+ and ER- subpopulations derived from the same patient. Single cell-derived colonies show that ER+ CTCs can produce ER- progeny, pointing to likely epigenetic mechanisms and underlying cell plasticity. We propose to investigate the epigenetic regulation of ER expression loss and its potential restoration, using genomic analyses combined with CRISPR functional screens. In Aim 1, we will compare chromatin landscapes of isogenic ER+ and ER- CTC subpopulations, define their distinct functional properties and identify factors that modulate their interconversion in vitro. In Aim 2, we will use undertake CRISPR screens to identify genes capable of restoring ER expression to CTC lines from HR+ breast cancers that have lost endogenous ER expression following endocrine therapy. Preliminary data indicate the feasibility of this approach, and we will explore mechanisms underlying restoration of ER expression, and whether this is accompanied by a return to ER-dependent proliferation. Together, these Aims address the plasticity of ER expression in patient-derived cultured CTCs that recapitulate clinical treatment exposures and tumor adaptation mechanisms. Loss of ER expression in HR + breast cancer may also lead to dependence on alternative oncogenic drivers, that may be constitute drug targets in refractory HR+ breast cancer. In Aim 3, we apply a chemical proteomic strategy in CTC lines, combining cysteine cross-linking with mass spectrometry to identify all ligandable cysteine residues within cellular proteins. Preliminary data show multiple cysteine-targetable proteins in CTCs that are not present in untreated breast cancer lines, and we will use CRISPR screens to identify those required for proliferation, followed by identification of tool compounds from a specialized library of cysteine-reactive covalent inhibitors for functional analyses. Together, these complementary approaches address the loss of ER expression in advanced HR+ breast cancers, with the goal of overcoming acquired resistance to endocrine therapy.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).