R-SPONDIN3 IS ASSOCIATED WITH BASAL-PROGENITOR BEHAVIOR IN NORMAL AND TUMOR MAMMARY CELLS

GARCÍA SOLA, MARTÍN E.; MEISS, ROBERTO P.; FELCHER, CARLA M.; ABBA, MARTÍN C.; TOCCI, JOHANNA M.; COSO, OMAR A.; KORDON, EDITH C.

Lucca

Conferencia; Gordon Research Conference Mammary Gland Development and Breast Cancer from Systems Biology to Single Cell; 2018

Gordon Research Conferences

R-spondin3 (RSPO3) has been implicated in several cancer models including colorectal, ovarian, and lung cancer. However, the role of this protein in breast cancer and in the maintenance of normal mammary stem cells (MaSCs) remains unclear. Here, we report that RSPO3 is co-expressed with mesenchymal and cancer stem cell (CSC) markers in breast cancer cells. In the normal mouse mammary gland, RSPO3 is expressed in the MaSC-enriched basal fraction and in the stroma, whilst it is absent from committed luminal epithelial cells. To validate the role of RSPO3 in the malignant phenotype, the mammary tumor cell line SCg6 was stably transfected with specific shRNA sequences to reduce expression of this protein. These cells showed morphological changes, increase of membrane-bound β-catenin, reduced expression of basal markers and a significant decrease in migration capacity. When inoculated into cleared fat pads of syngeneic mice, the Rspo3-KD cells generated smaller tumors, which arose later, showed lower expression of Ki67 and higher levels of cleaved-caspase 3 compared to controls. Besides, when inoculated into the tail vein, shRspo3 cells exhibited a dramatic reduction in their ability to develop experimental lung metastasis. In order to evaluate RSPO3 expression in human mammary tissue, we analyzed samples from 75 patients with ductal infiltrating breast carcinomas. We found that most of these samples showed positive immunoreactivity for RSPO3 (70%). In addition, the expression data of The Cancer Genome Atlas (TCGA) Network from 1985 human breast carcinoma samples, revealed that RSPO3 was highly expressed in triple negative samples (ER-PR-Her2-) compared to ER+ subtypes (ER+PR+/-Her2+/-), and showed significant RSPO3 overexpression in the basal-like subtype. Moreover, by sub-dividing basal-like breast cancer category within the intrinsic subtypes, we determined significantly high RSPO3 expression in claudin-low, basal-like tumors, which are characterized by low to absent expression of differentiated luminal cell markers, high enrichment for EMT markers, and CSC-like features. Taken together, these results suggest that RSPO3 could be relevant for breast cancer progression favoring the maintenance of a migratory basal/mesenchymal-like phenotype in mammary tumor cells. We hypothesize that blocking its activity might be important not only to reduce tumor bulk, but also to target the CSC population, which is responsible for metastatic spread and recurrence.