Abstract A048: Intracellular DKK1 could play a vital role in promoting triple-negative breast cancer cell proliferation, migration, and invasion

Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by extensive heterogeneity, high rates of metastasis and recurrence, and poor prognosis. Chemotherapy with severe toxicity still remains the mainstay systemic treatment. Although various molecular therapeutic targets exist for TNBC, the low proportion regarding the expression of those targets in TNBC tissues limits the number of the patients who benefit from the molecular therapeutics. Therefore, there is an urgent need to identify novel molecular targets those are expressed in the substantial proportion of TNBC patients. Methods: Dickkopf-1 (DKK1) protein expression within clinical TNBC tissues was evaluated using immunohistochemistry (IHC) assay, and the corresponding H-score was calculated. DKK1-positive TNBC tissues were defined by an H-score greater than 50 in the IHC analysis. As genetic approaches, DKK1 knockout MDA-MB-231 (231-DKK1 KO) / 4T1 (4T1-DKK1 KO) cell lines using CRISPR/Cas9 technology was constructed, respectively. As a pharmacologic approach, a single-strand nucleic acid aptamer specifically against DKK1 was then utilized to synthesize a DKK1 aptamer-based PROTAC (Apc102) with both exceptional cellular internalization ability and intracellular DKK1 degradation activity. The cellular internalization of DKK1 antibody and Apc102 was evaluated using fluorescence microscopy. Cell proliferative ability was evaluated using colony formation assay. Cell migratory and invasive abilities were evaluated using transwell assay. Results: In our clinical TNBC biobank (n=48), it was found that DKK1, an osteocyte-derived secretory protein, was positively expressed (H-score > 50) in a substantial proportion (73%) of clinical TNBC tissues. Bioinformatic analysis indicated that DKK1 expression was associated with poor prognosis in TNBC patients. DKK1 knockout significantly inhibited cell proliferation, migration, and invasion in both 231-DKK1 KO and 4T1-DKK1 KO in vitro. It suggested the vital role of DKK1 in TNBC. Anti-DKK1 antibody could not be internalized by TNBC cells and demonstrated no effects on cell proliferation, migration, and invasion in TNBC cells in vitro. It suggested that the role of extracellular DKK1 could be excluded in TNBC in vitro. Pharmacologically, Apc102 led to a significant inhibition of cell proliferation, migration, and invasion in MDA-MB-231 cell line in vitro. It suggested the vital role of intracellular DKK1 in TNBC. Conclusions: DKK1 was positively expressed in a substantial proportion of TNBC patients and associated with poor prognosis. Intracellular DKK1 could play a vital role in promoting TNBC cell proliferation, migration, and invasion.