The developmental receptor NOTCH plays an important role in various human cancers as a consequence of oncogenic mutations. Here we describe a novel mechanism of NOTCH-induced tumor suppression involving modulation of the deacetylase SIRT1, providing a rationale for the use of SIRT1 inhibitors to treat cancers where this mechanism is inactivated because of SIRT1 overexpression. In Ewing sarcoma cells, NOTCH signaling is abrogated by the driver oncogene EWS-FLI1. Restoration of NOTCH signaling caused growth arrest due to activation of the NOTCH effector HEY1, directly suppressing SIRT1 and thereby activating p53. This mechanism of tumor suppression was validated in Ewing sarcoma cells, B-cell tumors, and human keratinocytes where NOTCH dysregulation has been implicated pathogenically. Notably, the SIRT1/2 inhibitor Tenovin-6 killed Ewing sarcoma cells in vitro and prohibited tumor growth and spread in an established xenograft model in zebrafish. Using immunohistochemistry to analyze primary tissue specimens, we found that high SIRT1 expression was associated with Ewing sarcoma metastasis and poor prognosis. Our findings suggest a mechanistic rationale for the use of SIRT1 inhibitors being developed to treat metastatic disease in patients with Ewing sarcoma. Cancer Res; 74(22); 1–11. ©2014 AACR.

The developmental receptor NOTCH plays an important role in various human cancers as a consequence of oncogenic mutations. Here we describe a novel mechanism of NOTCH-induced tumor suppression involving modulation of the deacetylase SIRT1, providing a rationale for the use of SIRT1 inhibitors to treat cancers where this mechanism is inactivated due to SIRT1 overexpression. In Ewing sarcoma (ES) cells, NOTCH signaling was abrogated by the driver oncogene EWS-FLI1. Restoration of NOTCH signaling caused growth arrest due to activation of the NOTCH effector HEY1, directly suppressing SIRT1 and thereby activating p53. This mechanism of tumor suppression was validated in ES cells, B cell tumors and human keratinocytes where NOTCH dysregulation has been implicated pathogenically. Notably, the SIRT1/2 inhibitor Tenovin-6 killed ES cells in vitro and prohibited tumor growth and spread in an established xenograft model in zebrafish. Using immunohistochemistry to analyze primary tissue specimens, we found that high SIRT1 expression was associated with ES metastasis and poor prognosis. Our findings suggest a mechanistic rationale for the use of SIRT1 inhibitors being developed to treat metastatic disease in ES patients.

Age and physiologic status, such as menopause, are risk factors for breast cancer. Less clear is what factors influence the diversity of breast cancer. In this study, we investigated the effect of host age on the distribution of tumor subtypes in mouse mammary chimera consisting of wild-type hosts and Trp53 nullizygous epithelium, which undergoes a high rate of neoplastic transformation. Wild-type mammary glands cleared of endogenous epithelium at 3 weeks of age were subsequently transplanted during puberty (5 weeks) or at maturation (10 weeks) with syngeneic Trp53-null mammary tissue fragments and monitored for one year. Tumors arose sooner from adult hosts (AH) compared with juvenile hosts (JH). However, compared with AH tumors, JH tumors grew several times faster, were more perfused, exhibited a two-fold higher mitotic index, and were more highly positive for insulin-like growth factor receptor phosphorylation. Most tumors in each setting were estrogen receptor (ER)-positive (80% JH vs. 70% AH), but JH tumors were significantly more ER-immunoreactive (P = 0.0001) than AH tumors. A differential expression signature (JvA) of juvenile versus adult tumors revealed a luminal transcriptional program. Centroids of the human homologs of JvA genes showed that JH tumors were more like luminal A tumors and AH tumors were more like luminal B tumors. Hierarchical clustering with the JvA human ortholog gene list segregated luminal A and luminal B breast cancers across datasets. These data support the notion that age-associated host physiology greatly influences the intrinsic subtype of breast cancer. Cancer Res; 74(23); 1–10. ©2014 AACR.

Age and physiological status, like menopause, are risk factors for breast cancer. Less clear is what factors influence the diversity of breast cancer. In this study, we investigated the effect of host age on the distribution of tumor subtypes in mouse mammary chimera consisting of wild-type hosts and Trp53 nullizygous epithelium, which undergoes a high rate of neoplastic transformation. Wild-type mammary glands cleared of endogenous epithelium at 3 weeks of age were subsequently implanted during puberty (5 weeks) or at maturation (10 weeks) with syngeneic Trp53 null mammary tissue fragments and monitored for 1 year. Tumors arose sooner from adult hosts (AH) compared to juvenile hosts (JH). However, compared to AH tumors, JH tumors grew several times faster, were more perfused, exhibited a 2-fold higher mitotic index and were more highly positive for insulin-like growth factor receptor phosphorylation. Most tumors in each setting were ER positive (80% JH vs 70% AH) but JH tumors were significantly more ER immunoreactive (p=0.0001) than AH tumors. A differential expression signature (JvA) of juvenile versus adult tumors revealed a luminal transcriptional program. Centroids of the human homologs of JvA genes showed that JH tumors were more like luminal A tumors and AH tumors were more like luminal B tumors. Hierarchical clustering with the JvA human ortholog gene list segregated luminal A and luminal B breast cancers across data sets. These data support the notion that age-associated host physiology greatly influences the intrinsic subtype of breast cancer.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine tumor of the skin currently with no cure. In this study, we have first demonstrated that c-Myc overexpression is common in MCC. By targeting c-Myc, bromodomain inhibitors have demonstrated antitumor efficacy in several preclinical human cancer models. Thus, we interrogated the role of c-Myc inhibition in MCC with c-Myc amplification by using the BET inhibitor JQ1. We have uncovered that c-Myc can be regulated by JQ1 in MCC cells with pathologic c-Myc activation. Moreover, JQ1 potently abrogates c-Myc expression in MCC cells and causes marked G1 cell-cycle arrest. Mechanistically, JQ1-induced cell-cycle arrest coincides with downregulation of cyclin D1 and upregulation of p21, p27, and p57, whereas JQ1 exerts no effect on apoptosis in MCC cells. Further knockdown of p21, p27, or p57 by shRNA partially protects cells from JQ1-induced cell-cycle arrest. In addition, c-Myc knockdown by shRNA generates significant cell-cycle arrest, suggesting that c-Myc overexpression plays a role in MCC pathogenesis. Most importantly, JQ1 significantly attenuates tumor growth in xenograft MCC mouse models. Our results provide initial evidence, indicating the potential clinical utility of BET protein inhibitors in the treatment of MCC with pathologic activation of c-Myc. Cancer Res; 74(23); 1–13. ©2014 AACR.