INTRODUCTION: Basal-type, or triple-negative, breast cancer (lacking estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 expression) is a high-risk disease for which no molecular therapies are currently available. We studied genetic signatures of basal breast cancer potentially suitable for therapeutic intervention. METHODS: We analyzed protein expression of the Notch-1 intracellular domain and survivin by immunohistochemistry in a series of basal breast cancer patients. A hierarchical clustering and overall survival analysis was carried out on a microarray mRNA database of 232 breast cancer patients. Fifteen published mRNA datasets containing estrogen receptor-negative or estrogen receptor-positive samples were subjected to meta-analysis for co-segregated gene expression. Experiments of plasmid transfection and gene silencing were carried out in estrogen receptor-negative MDA-MB-231 breast cancer cells. RESULTS: The developmental signaling regulator Notch-1 was highly expressed in breast cancer, compared with normal tissue, and was segregated with basal disease. Higher Notch-1 levels correlated with progressively abbreviated overall survival, and with increased expression of survivin, a tumor-associated cell death and mitotic regulator implicated in stem cell viability. Analysis of Pearson's correlation coefficient indicated that Notch-1 and survivin co-segregated in basal breast cancer. Notch-1 stimulation in MDA-MB-231 cells increased survivin expression, whereas silencing Notch reduced survivin levels. CONCLUSIONS: A Notch-1-survivin functional gene signature is a hallmark of basal breast cancer, and may contribute to disease pathogenesis. Antagonists of Notch and survivin currently in the clinic may be tested as novel molecular therapy for these recurrence-prone patients.

Despite progress in the management of breast cancer, the molecular underpinnings of clinically aggressive subtypes of the disease are not well-understood. Here, we show that activation of Notch developmental signaling in estrogen receptor (ER)-negative breast cancer cells results in direct transcriptional up-regulation of the apoptosis inhibitor and cell cycle regulator survivin. This response is associated with increased expression of survivin at mitosis, enhanced cell proliferation, and heightened viability at cell division. Conversely, targeting Notch signaling with a peptidyl gamma-secretase inhibitor suppressed survivin levels, induced apoptosis, abolished colony formation in soft agar, and inhibited localized and metastatic tumor growth in mice, without organ or systemic toxicity. In contrast, ER+ breast cancer cells, or various normal cell types, were insensitive to Notch stimulation. Therefore, ER- breast cancer cells become dependent on Notch-survivin signaling for their maintenance, in vivo. Therapeutic targeting of this pathway may be explored for individualized treatment of patients with clinically aggressive, ER- breast cancer.

The 21st century brought about a dramatic increase in knowledge about genetic and molecular profiles of cancer. This information has validated the complexity of tumor cells and increased awareness of “nodal proteins”, but has yet to advance the development of rational targeted cancer therapeutics. Nodal proteins are critical cellular proteins that collect biological inputs and distribute the information across diverse biological processes. Survivin acts as a nodal protein by interfacing the multiple signals involved in mitosis and apoptosis and functionally integrate proliferation, cell death, and cellular homeostasis. By characterizing survivin as a target of both Type 1 Insulin-like Growth Factor (IGF-1) and Notch developmental signaling, we contribute to the paradigm of survivin as a nodal protein. The two signaling systems, Notch and IGF-1, regulate survivin by two independent mechanisms. Notch activation induces survivin transcription preferentially in basal breast cancer, a breast cancer subtype with poor prognosis and lack of molecular therapies. Activated Notch binds the transcription factor RBP-Jк and drives transcription from the survivin promoter. Notch mediated survivin expression increases cell cycle kinetics promoting tumor proliferation. Inhibition of Notch in a breast xenograft model reduced tumor growth and systemic metastasis. On the other hand, IGF-1 signaling drives survivin protein translation in prostate cancer cells. Binding of IGF-1 to its receptor activates downstream kinases, mammalian target of rapamycin (mTOR) and p70 S6 protein kinase (p70S6K), which modulates survivin mRNA translation to increase the apoptotic threshold. The multiple roles of survivin in tumorigenesis implicate survivin as a rational target for the “next generation” of cancer therapeutics.

Deregulated cell division is a hallmark of cancer, but whether tumor cells become dependent on specific mitotic mechanisms is not known. Here, we show that the small GTPase Ran, a regulator of mitotic spindle formation, is differentially overexpressed in human cancer as compared with normal tissues, in vivo. Acute silencing of Ran in various tumor cell types causes aberrant mitotic spindle formation, mitochondrial dysfunction, and apoptosis. This pathway does not require p53, Bax, or Smac, but is controlled by survivin as a novel Ran target in cancer. Conversely, loss of Ran in normal cells is well tolerated and does not result in mitotic defects or loss of cell viability. Therefore, tumor cells can become dependent on Ran signaling for cell division, and targeting this pathway may provide a novel and selective anticancer strategy.