Citations

Abstract Background: NOTCH oncogenic signaling induces epithelial-to-mesenchymal transition (EMT) and tumor stemness that play a critical role in driving tumor progression. Following interaction of NOTCH receptors (NOTCH 1, 2, 3, and 4) with their ligands (DELTA-LIKE 1, 3, 4 and JAGGED 1, 2), γ- secretase complex performs an intra-membrane cleavage releasing the NOTCH intracellular domain (NNICD). NNICD translocates to the nucleus and induces the expression of HEY1 and HES1 transcription factors that in turn orchestrate NOTCH-mediated EMT and stemness reprogramming. Materials and Methods: Parental MCF-7 and unique metastatic MCF-7RAF-1 xenografts with constitutively active MAPK signaling (D’Assoro et al., Oncogene 2014: 33:599-610) were used to define the extent to which increased expression of NOTCH signaling was linked to distant metastases. SKY analysis was performed to assess the grade of chromosomal instability (CIN) in tumor xenografts. Stemness activity was assessed by culturing breast cancer cells under non-adherent conditions to generate mammospheres. Expression of CD24 luminal marker was characterized by FACS analysis and immunofluorescence. Global transcriptome analysis was performed using Affymetrix® microarray. Highly invasive MDA-MB 231 breast cancer cells were used to perform a tail vein cancer cell-seeding assay. The LY411575 γ-secretase inhibitor (Pan-NOTCH inhibitor) was used to block NOTCH signaling in MDA-MB 231 cells. Results: Metastasis Initiating Cells (MICs) isolated from the lungs of nude mice carrying MCF-7RAF-1 xenografts exhibited a higher degree of genomic stability than primary tumor xenografts that was characterized by the absence of centrosome amplification and non-clonal chromosomal abnormalities. MICs also showed higher mammosphere forming capacity that was linked to lower expression of luminal marker CD24. Significantly, global transcriptome analysis showed that expression of NOTCH3 and HES1 stemness genes was increased in MICs. To corroborate the role of NOTCH signaling in inducing metastatic seeding, MDA-MB 231 breast cancer cells were pre-treated with 1 µM of LY411575 After 72 hours viable cells were injected into the tail vein of immune-compromised mice to develop lung metastases. While animals injected with untreated MDA-MB 231 cells developed lung metastases, those injected with the LY411575-treated MDA-MB 231 cells failed to develop lung metastatic lesions. Conclusions: These preliminary findings demonstrate the role of NOTCH stemness signaling in promoting metastatic seeding of breast cancer cells. Since NOTCH signaling is a “druggable target”, these results also provide the rationale to develop novel NOTCH-targeted therapies to inhibit tumor stemness and early onset of distant metastases. Citation Format: Alexey Leontovich, Jeffrey Salisbury, Candace L. Haddox, Lisa Mills, Tufia Haddad, James McCubrey, Matthew Goetz, James Ingle, Evanthia Galanis, Antonino B. D'Assoro. NOTCH signaling promotes metastatic seeding and growth of human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4851. doi:10.1158/1538-7445.AM2017-4851

Abstract Background: Intrinsic resistance has been linked to ER down-regulation and poor clinical outcome in breast tumors. Aurora-A kinase (AURKA) promotes distant metastases through activation of epithelial to mesenchymal transition (EMT) programming and expansion of breast tumor initiating cells (BTICs) harboring a CD44high/CD24low phenotype. Moreover, AURKA induces down-regulation of ERα expression and resistance to endocrine therapy that is mediated by SMAD5 transcription factor (D’Assoro et al., Oncogene 2014: 33:599-610). The aim of this study was to evaluate the role of AURKA in endocrine resistance induced by breast cancer stemness and ER down-regulation. Materials and Methods: We employed parental MCF-7 and variant MCF-7 cells ex-vivo (vMCF- 7/LCC9, vMCF-7/FR) that have high endogenous levels of AURKA, are ERlow, and exhibit in vivo resistance to fulvestrant. Real-Time Cell Proliferation and Apoptosis assays were performed to assess response to endocrine and/or AURKA-targeted therapy using the innovative IncuCyte ZOOM System. Stemness activity was assessed by culturing breast cancer cells under non-adherent conditions to generate mammospheres. Expression of ER, CD44 and CD24 markers was characterized by FACS analysis and immunofluorescence. Results: Pharmacologic inhibition of AURKA activity with the small molecule, alisertib, significantly restored sensitivity to fulvestrant in vMCF-7/LCC9 and vMCF-7/FR cells. To establish whether fulvestrant resistance was linked to enrichment of BTICs, breast cancer cells were cultured under non-adherent conditions to generate mammospheres. vMCF-7/FR cells generated mammospheres at higher efficiency compared to parental cells suggesting that vMCF-7/FR cells exhibited high self-renewal activity. vMCF-7/FR-derived mammospheres displayed a stem cell-like CD44high/CD24low phenotype that was further characterized by low levels of ER expression and resistance to fulvestrant. When vMCF-7 FR-derived mammospheres were treated with alisertib alone or combined with fulvestrant, the combination was more effective in mediating restoration of endocrine sensitivity. The phenotype of the alisertib-treated mammospheres was functionally linked to loss of CD44 expression, restoration of CD24 and ER expression, and sensitivity to fulvestrant. Conclusions: These findings provide further evidence that AURKA activity plays a central role in breast cancer progression through the genesis and clonal expansion of BTICs that are characterized by ER down-regulation and resistance to endocrine therapy. These results are important for the treatment of endocrine resistant breast tumors because they provide the rationale for the development of an innovative therapeutic approach to combine endocrine therapy with a targeted inhibitor of AURKA activity. Citation Format: Antonino B. D’Assoro, Tufia Haddad, Matthew Goetz, Mary Kuffel, John Hawse, Vera Suman, Jeffrey Salisbury, James Ingle, Evanthia Galanis. Pharmacologic targeting of Aurora-A kinase restores ERα expression and endocrine sensitivity through impairment of breast cancer stemness. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3313.

Abstract Breast cancer is the number one diagnosed cancer in women. Luminal breast cancers express steroid hormone receptors (SR) and these cases can be effectively treated with endocrine therapies that block estrogen receptor (ER) activity or estrogen synthesis. Unfortunately, at least 40% of women develop resistance to anti-estrogen treatments and progress to metastatic disease. One of the important transitions from hormone responsive (ER+/PR+ luminal A) to refractory (ER+ luminal B) disease involves the loss of progesterone receptor (PR) protein expression. However, data from large clinical trials showed that exposure to a synthetic progestin (MPA) and estrogen, but not estrogen alone, increased breast cancer incidence and tumor grade. These data, while somewhat controversial, implicate PR action in breast cancer development and progression. PR isoforms (A and B) are ligand-activated transcription factors, though they can be activated in the absence of progestin via signaling pathways commonly elicited downstream of growth factor receptors. We have shown that growth factor signaling (EGF or heregulin) through PR-B can initiate rapid proliferation and survival of breast cancer cells growing in soft-agar. PR-B is phosphorylated and hyperactived by mitogenic protein kinases, including MAPK, CDK2, and CK2. Notably, over-expression and activation of Aurora A kinase (downstream of MAPKs) is associated with invasive breast cancer and poor prognosis. We hypothesize that activation of protein kinases commonly overexpressed in breast cancer provide a context for aberrant PR activity, resulting in upregulation of Her2/erbB2 signaling, EMT, and increased expression of mammary stem-cell mediators (stemness). To model this process, we over-expressed oncogenic Raf1 in ER+/PR+ MCF-7 breast cancer cells. MCF-7-Raf1 cells exhibited constitutive activation of the MAPK pathway and upregulation of both ER and PR mRNA and protein relative to parental controls. Addition of progestin to MCF-7-Raf1 cells increased PR phosphorylation and soft agar growth that was blocked by MLN8237, an Aurora kinase inhibitor. Interestingly, passage of MCF-7-Raf1 cells as mouse xenografts (1GX) led to rapid tumor progression that was associated with increased Aurora A phosphorylation, loss of PR mRNA and protein, expression of epithelial-to-mesenchymal (EMT) markers, increased stemness, and tamoxifen resistance. These data suggest that Aurora A kinase and PR cross-talk may drive early breast cancer progression in response to growth promoting signals. Targeting this cross-talk with Aurora kinase inhibitors and anti-progestins, in conjunction with conventional estrogen-blocking therapies, may improve survival outcomes by preventing progression to endocrine failure. MCF-7-Raf1 and MCF-7-Raf-1 (1GX) cells may provide a useful model for the study of SR-loss during luminal A to luminal B transition. Citation Format: Katherine A. Leehy, Tarah M. Regan Anderson, Andrea R. Daniel, Antonino B. D'Assoro, Carol A. Lange. Aurora A kinase and progesterone receptor cross talk in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2107. doi:10.1158/1538-7445.AM2014-2107

New-onset diabetes mellitus after transplantation (NODAT) may complicate 2–50% of kidney transplantation, and it is associated with reduced graft and patient survivals. In this retrospective study, we applied a conversion protocol to sirolimus in a cohort of kidney transplant recipients with NODAT. Among 344 kidney transplant recipients, 29 patients developed a NODAT (6.6%) and continued with a reduced dose of calcineurin inhibitors (CNI) (8 patients, Group A) or were converted to sirolimus (SIR) (21 patients, Group B). NODAT resolved in 37.5% and in 80% patients in Group A and Group B, respectively. In Group A, patient and graft survivals were 100% and 75%, respectively, not significantly different from Group B (83.4% and 68%, resp.,P=0.847). Graft function improved after conversion to sirolimus therapy: serum creatinine was 1.8 ± 0.7 mg/dL at the time of conversion and 1.6 ± 0.4 mg/dL five years after conversion to sirolimus therapy (P<0.05), while in the group of patients remaining with a reduced dose of CNI, serum creatinine was 1.7 ± 0.6 mg/dL at the time of conversion and 1.65 ± 0.6 mg/dL at five-year followup (P=0.732). This study demonstrated that the conversion from CNI to SIR in patients could improve significantly the metabolic parameters of patients with NODAT, without increasing the risk of acute graft rejection.

Abstract In this study we demonstrate that gain of HER-2/Neu expression in human breast cancer xenografts harboring constitutive active Raf-1 induces Aurora-A over-expression that ultimately drives the transition from a luminal to a highly invasive basal-like phenotype. Over-expression of Aurora-A was induced by phosphorylation and protein stabilization by HER-2/Neu rather than increased Aurora-A gene expression. Moreover, Aurora-A stabilization induced an increase of HER-2/Neu expression in breast cancer cells defining a novel positive feed-back loop between Aurora-A and HER-2/Neu oncogenic signaling pathways. Basal-like tumor cells over-expressing Aurora-A and HER-2/Neu underwent to loss of CD24 surface receptor, expressed epithelial to mesenchymal transition (EMT) markers and acquired stem cell-like properties such as mammospheres formation and self-renewal. Pharmacologic inhibition of HER-2/Neu signaling pathway with lapatinib in cancer cells over-expressing Aurora-A demonstrated that Aurora-A was downstream to HER-2/Neu in the development of EMT and stemness. To determine whether Aurora-A induced a cancer stem cell-like phenotype through development of chromosomal instability and tumor cell phenotypic heterogeneity, we performed a SKY analysis on the breast cancer cell lines employed in this study. Our results demonstrate that invasive breast cancer cells harboring a basal-like phenotype did not develop higher chromosomal instability compared to luminal non-invasive cancer cells, signifying that Aurora-A has a direct effect on the development of basal-like breast cancer cells with stemness properties. Importantly, pharmacologic inhibition of Aurora-A kinase activity with the small molecule MLN8237 restored CD24 expression and reversed EMT leading to the re-establishment of a luminal phenotype and suppression of distant metastases. Our findings implicate for the first time the mitotic kinase Aurora-A in the development and maintenance of CD24 negative basal-like tumor stem cells responsible for breast cancer recurrence and progression. These observations have also important implications for the development of molecular targeted therapies to inhibit Aurora-A kinase activity and stemness improving the overall survival of breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 328. doi:1538-7445.AM2012-328