NOTCH3 expression is linked to breast cancer seeding and distant metastasis.

BACKGROUND: Development of distant metastases involves a complex multistep biological process termed the invasion-metastasis cascade, which includes dissemination of cancer cells from the primary tumor to secondary organs. NOTCH developmental signaling plays a critical role in promoting epithelial-to-mesenchymal transition, tumor stemness, and metastasis. Although all four NOTCH receptors show oncogenic properties, the unique role of each of these receptors in the sequential stepwise events that typify the invasion-metastasis cascade remains elusive. METHODS: We have established metastatic xenografts expressing high endogenous levels of NOTCH3 using estrogen receptor alpha-positive (ERα+) MCF-7 breast cancer cells with constitutive active Raf-1/mitogen-associated protein kinase (MAPK) signaling (vMCF-7Raf-1) and MDA-MB-231 triple-negative breast cancer (TNBC) cells. The critical role of NOTCH3 in inducing an invasive phenotype and poor outcome was corroborated in unique TNBC cells resulting from a patient-derived brain metastasis (TNBC-M25) and in publicly available claudin-low breast tumor specimens collected from participants in the Molecular Taxonomy of Breast Cancer International Consortium database. RESULTS: In this study, we identified an association between NOTCH3 expression and development of metastases in ERα+ and TNBC models. ERα+ breast tumor xenografts with a constitutive active Raf-1/MAPK signaling developed spontaneous lung metastases through the clonal expansion of cancer cells expressing a NOTCH3 reprogramming network. Abrogation of NOTCH3 expression significantly reduced the self-renewal and invasive capacity of ex vivo breast cancer cells, restoring a luminal CD44low/CD24high/ERαhigh phenotype. Forced expression of the mitotic Aurora kinase A (AURKA), which promotes breast cancer metastases, failed to restore the invasive capacity of NOTCH3-null cells, demonstrating that NOTCH3 expression is required for an invasive phenotype. Likewise, pharmacologic inhibition of NOTCH signaling also impaired TNBC cell seeding and metastatic growth. Significantly, the role of aberrant NOTCH3 expression in promoting tumor self-renewal, invasiveness, and poor outcome was corroborated in unique TNBC cells from a patient-derived brain metastasis and in publicly available claudin-low breast tumor specimens. CONCLUSIONS: These findings demonstrate the key role of NOTCH3 oncogenic signaling in the genesis of breast cancer metastasis and provide a compelling preclinical rationale for the design of novel therapeutic strategies that will selectively target NOTCH3 to halt metastatic seeding and to improve the clinical outcomes of patients with breast cancer.

Inhibition of Cdk2 kinase activity selectively targets the CD44⁺/CD24⁻/Low stem-like subpopulation and restores chemosensitivity of SUM149PT triple-negative breast cancer cells.

Inflammatory breast cancer (IBC) is an angioinvasive and most aggressive type of advanced breast cancer characterized by rapid proliferation, chemoresistance, early metastatic development and poor prognosis. IBC tumors display a triple-negative breast cancer (TNBC) phenotype characterized by centrosome amplification, high grade of chromosomal instability (CIN) and low levels of expression of estrogen receptor α (ERα), progesterone receptor (PR) and HER-2 tyrosine kinase receptor. Since the TNBC cells lack these receptors necessary to promote tumor growth, common treatments such as endocrine therapy and molecular targeting of HER-2 receptor are ineffective for this subtype of breast cancer. To date, not a single targeted therapy has been approved for non-inflammatory and inflammatory TNBC tumors and combination of conventional cytotoxic chemotherapeutic agents remains the standard therapy. IBC tumors generally display activation of epithelial to mesenchymal transition (EMT) that is functionally linked to a CD44+/CD24-/Low stem-like phenotype. Development of EMT and consequent activation of stemness programming is responsible for invasion, tumor self-renewal and drug resistance leading to breast cancer progression, distant metastases and poor prognosis. In this study, we employed the luminal ER+ MCF-7 and the IBC SUM149PT breast cancer cell lines to establish the extent to which high grade of CIN and chemoresistance were mechanistically linked to the enrichment of CD44+/CD24low/- CSCs. Here, we demonstrate that SUM149PT cells displayed higher CIN than MCF-7 cells characterized by higher percentage of structural and numerical chromosomal aberrations. Moreover, centrosome amplification, cyclin E overexpression and phosphorylation of retinoblastoma (Rb) were restricted to the stem-like CD44+/CD24-/Low subpopulation isolated from SUM149PT cells. Significantly, CD44+/CD24-/Low CSCs displayed resistance to conventional chemotherapy but higher sensitivity to SU9516, a specific cyclin-dependent kinase 2 (Cdk2) inhibitor, demonstrating that aberrant activation of cyclin E/Cdk2 oncogenic signaling is essential for the maintenance and expansion of CD44+/CD24-/Low CSC subpopulation in IBC. In conclusion, our findings propose a novel therapeutic approach to restore chemosensitivity and delay recurrence of IBC tumors based on the combination of conventional chemotherapy with small molecule inhibitors of the Cdk2 cell cycle kinase.

Raf-1 oncogenic signaling is linked to activation of mesenchymal to epithelial transition pathway in metastatic breast cancer cells.

Aberrant activation of the Raf/MEK/MAPK pathway plays a key role in breast cancer development and progression. Dysregulation of Raf/MEK/MAPK oncogenic signaling often results from overexpression of the HER-2/Neu tyrosine kinase receptor leading to chemoendocrine resistance, development of distant metastases and ultimately poor prognosis in breast cancer patients. HER-2/Neu overexpression is also linked to activation of the epithelial to mesenchymal transition (EMT) pathway, loss of adhesion molecules and metastasis. Recently, it has been demonstrated that cancer cells that undergo EMT acquire a CD44+/CD24-/low basal cancer stem cell-like phenotype and are characterized by activation of HER-2/Neu and TGFß oncogenic signaling pathways with increased capacity of self-renewal, drug resistance, invasion and distant metastases. Following metastatic dissemination, cancer cells re-activate certain epithelial properties through mesenchymal to epithelial transition (MET) to establish neoplastic lesions at secondary sites, although the molecular mechanisms regulating MET remain elusive. In this study we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces HER-2/Neu overexpression leading to the development of distant metastases in ERα+ MCF-7 breast cancer xenografts. Importantly, development of distant metastases in xenograft models was linked to activation of the MET pathway characterized by reduced expression of EMT inducer genes (TGFB2, TWIST1 and FOXC1) and overexpression of BMB7, CXCR7 and EGR family of transcription factors. In summary, our results demonstrate for the first time that amplification of Raf/MEK/MAPK oncogenic signaling during tumor growth promotes the genesis of metastatic lesions from primary tumors by activating the mesenchymal epithelial transition.