Spyros Stylianou

Aberrant Activation of Notch Signaling in Human Breast Cancer

A role for Notch signaling in human breast cancer has been suggested by both the development of adenocarcinomas in the murine mammary gland following pathway activation and the loss of Numb expression, a negative regulator of the Notch pathway, in a large proportion of breast carcinomas. However, it is not clear currently whether Notch signaling is frequently activated in breast tumors, and how it causes cellular transformation. Here, we show accumulation of the intracellular domain of Notch1 and hence increased Notch signaling in a wide variety of human breast carcinomas. In addition, we show that increased RBP-Jkappa-dependent Notch signaling is sufficient to transform normal breast epithelial cells and that the mechanism of transformation is most likely through the suppression of apoptosis. More significantly, we show that attenuation of Notch signaling reverts the transformed phenotype of human breast cancer cell lines, suggesting that inhibition of Notch signaling may be a therapeutic strategy for this disease.

Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor.

Notch receptor signaling pathways play an important role not only in normal breast development but also in breast cancer development and progression. We assessed the role of Notch receptors in stem cell activity in breast cancer cell lines and nine primary human tumor samples. Stem cells were enriched by selection of anoikis-resistant cells or cells expressing the membrane phenotype ESA(+)/CD44(+)/CD24(low). Using these breast cancer stem cell populations, we compared the activation status of Notch receptors with the status in luminally differentiated cells, and we evaluated the consequences of pathway inhibition in vitro and in vivo. We found that Notch4 signaling activity was 8-fold higher in stem cell-enriched cell populations compared with differentiated cells, whereas Notch1 signaling activity was 4-fold lower in the stem cell-enriched cell populations. Pharmacologic or genetic inhibition of Notch1 or Notch4 reduced stem cell activity in vitro and reduced tumor formation in vivo, but Notch4 inhibition produced a more robust effect with a complete inhibition of tumor initiation observed. Our findings suggest that Notch4-targeted therapies will be more effective than targeting Notch1 in suppressing breast cancer recurrence, as it is initiated by breast cancer stem cells.

Notch Activation Induces Akt Signaling via an Autocrine Loop to Prevent Apoptosis in Breast Epithelial Cells

The Notch pathway is aberrantly activated in a wide range of cancers, including breast carcinoma, and is required to maintain the transformed phenotype of many of these tumors. Notch signaling contributes to the transformed phenotype, in part, by preventing apoptosis in response to many different stimuli. However, it is unclear how Notch activation can lead to a general suppression of apoptosis. We show here that Notch signaling induced an autocrine signaling loop that activates Akt in breast epithelial cells. This activation of Akt was necessary for Notch-induced protection against apoptosis in the nontransformed breast epithelial cell line MCF10A. Moreover, inhibiting Notch signaling in breast cancer cells induced a decrease in Akt activity and an increase in sensitivity to apoptosis. Finally, the inhibition of ASK1 by Akt was responsible for the protection from apoptosis induced by DNA damage, as it prevented c-Jun NH(2)-terminal kinase-mediated phosphorylation and activation of p53.

Aberrant activation of Notch signalling in human breast cancer

A role for Notch signaling in human breast cancer has been suggested by both the development of adenocarcinomas in the murine mammary gland following pathway activation and the loss of Numb expression, a negative regulator of the Notch pathway, in a large proportion of breast carcinomas. However, it is not clear currently whether Notch signaling is frequently activated in breast tumors, and how it causes cellular transformation. Here, we show accumulation of the intracellular domain of Notch1 and hence increased Notch signaling in a wide variety of human breast carcinomas. In addition, we show that increased RBP-Jkappa-dependent Notch signaling is sufficient to transform normal breast epithelial cells and that the mechanism of transformation is most likely through the suppression of apoptosis. More significantly, we show that attenuation of Notch signaling reverts the transformed phenotype of human breast cancer cell lines, suggesting that inhibition of Notch signaling may be a therapeutic strategy for this disease.

Abstract 234: Inhibition of the NOTCH transcription factor complex by a novel hybrid protein leads to effective tumor therapy.

The Notch pathway has been implicated in the generation and propagation of cancer stem cells and is thus an attractive target for therapy. We have generated a hybrid protein (Antp-DNMAML) consisting of the truncated version of Mastermind‐like (MAML) that behaves in a dominant negative (DN) fashion inhibiting Notch, and the cell penetrating peptide Antennapedia (Antp). Results indicate that the Antp-DNMAML translocates into the nucleus, suppresses Notch activation, reverts the transformed phenotype, inhibits the anchorage‐dependent growth and induces self contact inhibition and apoptosis in tumorigenic human breast cancer cells. More significantly, there is direct evidence that inhibiting Notch signaling at the transcriptional level with the Antp-DNMAML protein, suppresses the expression of downstream Notch targets and inhibits tumor growth in nude mice, without organ or systemic toxicity. In summary, intracellular delivery of dominant-negative transcription complex proteins using the Antp platform is a new and specific approach for cancer therapy . Citation Format: Agamemnon A. Epenetos, Christina Kousparou, Spyros Stylianou, Mahendra Deonarain, Aleksandra Filipovic. Inhibition of the NOTCH transcription factor complex by a novel hybrid protein leads to effective tumor therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 234. doi:10.1158/1538-7445.AM2013-234

02-P017 Notch signalling in DCIS and invasive breast cancer

by a short BrdU pulse, indicating that prevention of Bmi1 downregulation does not drive granule cells back into the cell cycle. No tumours were observed in two aging cohorts of transgenic mice derived from separate founder lines. However, these mice display an increase in astrocytes with the type II morphology in vitro compared to wild-type littermates. Cultures of FACSorted Math1-GFP+ granule cells showed that the increase in astrocytes is not due to transdifferentiation of granule cells, but likely due to a cell extrinsic effect exerted by granule cells on glial progenitors in the cerebellum. Overexpression of Bmi1 in GCPs under the Math1 enhancer is also not sufficient to drive medulloblastoma formation. On the contrary, a reduction in cerebellar weight was observed. Abnormalities in cell death and cell proliferation are being investigated in these mice. In conclusion: high-level Bmi1 expression in postmitotic granule cells or in GCPs is not sufficient to initiate medulloblastoma development.