The molecular classification for breast carcinomas has been used in clinical studies with a simple surrogate panel of immunohistochemistry (IHC) markers. The objective of this current project was to study the molecular classification of commonly used breast cancer cell lines by IHC analysis. Seventeen breast cancer cell lines were harvested, fixed in formalin and made into cell blocks. IHC analyses were performed on each cell block with antibodies to estrogen receptor (ER), progesterone receptor (PR), HER2, EGFR, CK5/6, Ki-67 and androgen receptor (AR). Among the 17 cell lines, MCF-7 and ZR-75-1 fell to Luminal A subtype; BT-474 to Luminal B subtype; SKBR-3, MDA-MD-435 and AU 565 to HER2 over-expression subtype; MDA-MB-231, MCF-12A, HBL 101, HS 598 T, MCF-10A, MCF-10F, BT-20, 468 and BT-483 to basal subtype. MDA-MB-453 belonged to Unclassified subtype. Since each subtype defined by this IHC-based molecular classification does show a distinct clinical outcome, attention should be paid when choosing a cell line for any study.

Advanced breast cancers preferentially metastasize to bone where cells in the bone microenvironment produce factors that enhance breast cancer cell homing and growth. Expression of the ubiquitin E3 ligase WWP1 is increased in some breast cancers, but its role in bone metastasis has not been investigated. Here, we studied the effects of WWP1 and itch, its closest family member, on breast cancer bone metastasis. First, we immunostained a multi-tumor tissue microarray and a breast cancer tissue microarray and demonstrated that WWP1 and ITCH are expressed in some of breast cancer cases. We then knocked down WWP1 or itch in MDA-MB-231 breast cancer cells using shRNA and inoculated these cells and control cells into the left ventricle of athymic nude mice. Radiographs showed that mice given shWWP1 cells had more osteolytic lesions than mice given control MDA-MB-231 cells. Histologic analysis confirmed osteolysis and showed significantly increased tumor area in bone marrow of the mice. WWP1 knockdown did not affect cell growth, survival or osteoclastogenic potential, but markedly increased cell migration toward a CXCL12 gradient in vitro. Furthermore, WWP1 knockdown significantly reduced CXCL12-induced CXCR4 lysosomal trafficking and degradation. In contrast, itch knockdown had no effect on MDA-MB-231 cell bone metastasis. Taken together, these findings demonstrate that WWP1 negatively regulates cell migration to CXCL12 by limiting CXCR4 degradation to promote breast cancer metastasis to bone and highlight the potential utility of WWP1 as a prognostic indicator for breast cancer bone metastasis.

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