Citations

e13516 Background: Breast cancer affects thousands of women each year. Epithelial to Mesenchymal transition (EMT) has been associated with increased metastatic potential of cancer cells as well as resistance to chemotherapy. Predicatively, presence of EMT leads to worse prognosis. We have recently showed that Aurora-A plays a key role in development of EMT and increased ability of breast cancer cells for self renewal. Therefore we hypothesized that inhibition of Aurora-A will lead to increased sensitivity to chemotherapy. Methods: The hypothesis was tested in vitro. Cellular proliferation was tested using MTT assay and protein levels were determined on Western blots. Results: In ER+ cell line, MCF7, constitutive activation of Raf-1/MAPK signaling pathway leads to aberrant activation of Aurora-A kinase activity. These cells have decreased sensitivity to treatment with paclitaxel when compared to parental MCF7 cells. This effect is even more pronounced with over-expression of Aurora-A. The sensitivity to paclitaxel was restored with inhibition of Aurora-A by novel inhibitor, Alisertib. This was further explored in triple negative cells (MDA-MB-231) and cells expressing Her-2/neu (BT474) which express higher levels of Aurora-A. Combination of Alisertib and paclitaxel was superior when compared to either therapy alone. Similar effect was seen with use of anthracyclines. Inhibition of Aurora-A resulted in decreased SMAD5 expression as well as decreased Akt phosphorylation. Current studies are investigating a role of Aurora-A in developing chemoresistance through activating SMAD5. In vivo experiments evaluating combination therapies in breast cancer animal model are ongoing. Conclusions: Inhibition of Aurora-A by Alisertib resulted in decreased SMAD5 nuclear phosphorylation and increased effectiveness of chemotherapeutic agents in breast cancer cells. These results contribute to better understanding of signaling pathways involved in resistance of breast cancer cells to chemotherapy. This knowledge could be extremely useful in developing more effective treatments for breast cancer patients both in neo-adjuvant, adjuvant and metastatic settings.

e12123 Background: Onset of metastases involves a complex multistep biological process termed the invasion-metastasis cascade, which include dissemination of cancer cells from the primary tumor to secondary organs. NOTCH signaling plays a critical role in promoting Triple Negative Breast Cancer (TNBC) 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 established unique TNBC cells (TNBC-M14, -M25 and –M40), isolated from patient-derived brain metastasis xenografts (PD-BMXs). NOTCH3 genetic targeting was achieved using Lenti-vector shRNAs. Immunoblot analysis was used to assess NOTCH3 expression. ALDH1 activity was measured using the ALDEFLUOR Kit. To assess self-renewal capacity and resistance to docetaxel-based chemotherapy, TNBC cells were cultured under non-adherent conditions to form mammospheres (MPS). A publicly clinical database ( http://kmplot.com ) was employed to analyze NOTCH3 expression in a selected cohort of 107 lymph- node+ TNBC patients. Results: TNBC-M14, -M25 and -M40 MPS showed NOTCH3 overexpression and higher ALDH1 activity compared to MDA-MB 231 MPS used as control. To investigate the causative role of NOTCH3 in inducing ALDH1 activity, self-renewal capacity and chemoresistance, we infected TNBC MPS with Lenti-vector shRNAs targeting NOTCH3. NOTCH3 geneting targeting induced a significant reduction of ALDH1 activity compared to TNBC MPS infected with scrambled Lenti-shRNAs. NOTCH3 geneting targeting significantly impaired MPS formation and restored sensitivity to docetaxel. The correlation between aberrant NOTCH3 expression and poor outcome in patients with advanced TNBC was validated in a publicly clinical database. Conclusions: These findings provide a compelling preclinical rationale for the design of novel clinical trials that will selectively target NOTCH3 to restore chemosensitivity and to improve the progression-free survival of TNBC patients that lack FDA-approved targeted therapies.

Abstract Background: Aurora A (AURKA) is a mitotic kinase responsible for centrosome segregation and mitotic spindle formation. In normal cells, expression of AURKA is highly regulated and is predominantly restricted to G2/M phases of the cell cycle. Unlike healthy cells, cancer cells overexpress AURKA through all phases of the cell cycle resulting in the acquisition of alternate non-mitotic functions. Little is known about cellular functions regulated by AURKA and its interaction with other signaling molecules. Here, we report a novel interaction between AURKA and the mitogen-activated protein kinase (MAPK) pathway at the level of MEK1 in breast cancer cells. This interaction may serve as a novel target as well as demonstrate by an additive cytotoxic effect of AURKA- and MEK1/2-specific inhibitors against estrogen positive (ER+) and triple negative breast cancer (TNBC) cells. Results: We show that treatment of ER+ HER2- MCF-7, ER- HER2+ SKBR3 and ER- HER2- BT549 cells with AURKA specific inhibitors alisertib, MK8745 and Aurora A Inhibitor I resulted in over 2-fold increase in relative levels of poMEK1/2 and poERK1/2 compared to untreated controls. The activation of the MAPK pathway was rapid with changes seen within 5 min after treatment with AURKA inhibitors and was sustained for at least 48 hours. Treatment with the pan RAF inhibitor TAK-632 did not diminish alisertib-induced poERK and poMEK1/2. Alternatively, treatment with the MEK1/2 specific inhibitor PD0325901 completely abrogated alisertib-induced phosphorylation of MEK1/2 and ERK1/2. In situ proximity ligation and pull down assays demonstrated AURKA and MEK1/2 direct interaction. In vitro kinase assay showed direct phosphorylation of MEK1 by AURKA. Combined treatment of alisertib and PD0325901 in vitro revealed significant additive cytotoxic effect in MCF-7 and BT549 cells when compared to either agent used alone (p< 0.008 and p<0.011; p <0.04 and p<0.028) with early trend toward significance in survival in a BT549 xenograft breast cancer in vivo model. Conclusions: Our data shows a novel AURKA-MEK1 interaction in breast cancer cells. In depth in vivo analysis is ongoing. The results reveal a promising new strategy for the treatment TNBC patients using a combination of AURKA and MEK1/2 inhibitors. Citation Format: Gandhi S, Gil M, Khoury T, Takabe K, Puzanov I, Gelman I, D'Assoro A, Opyrchal M. A novel interaction of AURKA with MAPK pathway in breast cancer cells as a potential therapeutic target [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-17.

Abstract Background: Aurora-A (AURKA) belongs to the family of serine-threonine kinases that are crucial for cell cycle control. In particular, AURKA controls centrosome function, mitotic spindle formation, and proper chromosome segregation. In contrast to the well-defined function of AURKA in normal cells, the role of AURKA in malignant transformation and cancer progression is still under investigation. Here, we report that AURKA directly interacts with MEK1/2 kinase and induces chemotaxis of breast cancer cells. Results: We demonstrated that overexpression of AURKA in luminal ER+ MCF-7 cells (MCF-7AURKA) resulted in over 10-fold increase in the number of cells displaying chemotactic response upon stimulation with growth factors when compared to parental MCF-7 cells. Both AURKA inhibitor MLN8237 and MEK1/2 inhibitor PD0325901 reduced migratory properties of MCF-7AURKA cells. In contrast, treatment with the pan RAF inhibitor TAK-632 did not affect chemotactic response of MCF-7AURKA cells. We demonstrated colocalization of AURKA and MEK1/2 immunofluorescence signals in MCF-7AURKA and triple-negative BT549 cells. In situ proximity ligation assay (PLA) revealed the presence of AURKA-MEK1/2 complexes in MCF-7AURKA and BT549 cells, and the notion that AURKA and MEK1/2 formed cellular complexes was confirmed with co-immunoprecipitation experiments. In vitro kinase assays with recombinant AURKA and MEK1 proteins showed that AURKA kinase could directly phosphorylate MEK1. Incubation of MCF-7AURKA and BT549 cells with the AURKA-specific inhibitors MLN8237 or MK8745 resulted in over a 2-fold increase in the level of pERK1/2 compared to the untreated controls. Treatment with the pan RAF inhibitor TAK-632 did not diminish MLN8237-induced pERK1/2, whereas treatment with the MEK1/2 specific inhibitor PD0325901 completely abrogated MLN8237-induced activation of ERK1/2. Conclusions: Our data present a novel AURKA-MEK1/2 interaction that contributes to the increased chemotaxis of breast cancer cells. The results suggest that AURKA negatively regulates MEK1 through a novel, direct phosphorylation event, possibly leading to constitutive activation of MEK2 and increased chemotaxis. The combined treatment of AURKA and MEK1/2 inhibitors represents a new therapeutic approach to decrease metastatic potential of breast cancer cells and delay tumor progression. The in-depth analysis of the AURKA-MEK1/2 complexes as well as in vivo studies testing the efficacy of combining AURKA and MEK1/2 inhibitors on patient-derived xenografts are currently in progress. Citation Format: Malgorzata Gil, Archana Chidambaram, Thaer Khoury, Kazuaki Takabe, Igor Puzanov, Irwin H. Gelman, Antonino B. D’Assoro, Evanthia Galanis, Mateusz Opyrchal. AURKA interaction with MEK1/2 complex and its role in promoting breast cancer cell metastasis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A60.

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: Aurora A (AURKA) is a mitotic kinase responsible for centrosome segregation and mitotic spindle formation. In normal cells, expression of AURKA is highly regulated and is predominantly restricted to G2/M phases of the cell cycle. Unlike healthy cells, cancer cells overexpress AURKA through all phases of the cell cycle resulting in the acquisition of alternate non-mitotic functions. Little is known about cellular functions regulated by AURKA and its interaction with other signaling molecules. Here, we report a novel interaction between AURKA and the mitogen-activated protein kinase (MAPK) pathway in wild type BRAF breast cancer cells as well as demonstrate an additive cytotoxic effect of AURKA- and MEK1/2-specific inhibitors against estrogen positive (ER+) and triple negative (ER-, PR-, HER2-) breast cancer cells. Results: We show that treatment of ER+ HER2- MCF-7, ER- HER2+ SKBR3 and ER- HER2- BT549 cells with AURKA specific inhibitors alisertib, MK8745 and Aurora A Inhibitor I resulted in over 2-fold increase in the levels of both pMEK1/2 and pERK1/2 compared to the untreated controls. The activation of the MAPK pathway was rapid with changes seen within 5 min after treatment with AURKA inhibitors and was sustained for at least 48 hours. No differences in phosphorylation of MEK1/2 or ERK1/2 were observed in BRAFG464V triple negative MDA-MB-231 cells. Treatment with AURKA inhibitors resulted in downregulation of pAURKA and a significant increase in levels of total AURKA protein. The pull-down assay with Ras-binding domain coated agarose beads followed by western blot analysis with anti-pan-RAS Ab revealed no changes in active GTP-bound RAS in alisertib-treated MCF-7 cells compared to the untreated control. Consistently, no significant changes were observed in RAS-inducible phosphorylation of RAF1 activation site at Ser338 as demonstrated by western blot. Treatment with the pan RAF inhibitor TAK-632 did not diminish alisertib-induced pERK and pMEK1/2. Alternatively, treatment with the MEK1/2 specific inhibitor PD0325901 completely abrogated alisertib-induced phosphorylation of MEK1/2 and ERK1/2. Furthermore, combined treatment of alisertib and PD0325901 in vitro revealed significant additive cytotoxic effect in MCF-7 and BT549 cells when compared to either agent used alone (p< 0.008 and p<0.011; p <0.04 and p<0.028). Conclusions: Our data suggests that AURKA is a RAF1-independent negative regulator of MAPK activity in breast cancer cells. The in-depth analysis of the AURKA-MEK1/2 interaction is currently under investigation. The results reveal a promising new strategy for the treatment of wild type BRAF, TNBC patients using a combination of AURKA and MEK1/2 inhibitors. Citation Format: Malgorzata Gil, Archana Chidambaram, Thaer Khoury, Kazuaki Takabe, Igor Puzanov, Irwin Gelman, Antonio D’Assoro, Mateusz Opyrchal. Inhibition of AURKA induces Raf1-independent activation of MAPK pathway in 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 2123. doi:10.1158/1538-7445.AM2017-2123