Xuemei Xie

MEK Inhibitor Selumetinib (AZD6244; ARRY-142886) Prevents Lung Metastasis in a Triple-Negative Breast Cancer Xenograft Model

Patients with triple-negative breast cancer (TNBC) have a poor prognosis because TNBC often metastasizes, leading to death. Among patients with TNBC, those with extracellular signal-regulated kinase 2 (ERK2)-overexpressing tumors were at higher risk of death than those with low-ERK2-expressing tumors (hazard ratio, 2.76; 95% confidence interval, 1.19–6.41). The MAPK pathway has been shown to be a marker of breast cancer metastasis, but has not been explored as a potential therapeutic target for preventing TNBC metastasis. Interestingly, when we treated TNBC cells with the allosteric MEK inhibitor selumetinib, cell viability was not reduced in two-dimensional culture. However, in three-dimensional culture, selumetinib changed the mesenchymal phenotype of TNBC cells to an epithelial phenotype. Cells that undergo epithelial–mesenchymal transition (EMT) are thought to contribute to the metastatic process. EMT leads to generation of mesenchymal-like breast cancer cells with stem cell–like characteristics and a CD44+CD24−/low expression pattern. We tested the hypothesis that targeted inhibition of the MAPK pathway by selumetinib inhibits acquisition of the breast cancer stem cell phenotype and prevents lung metastasis of TNBC. TNBC cells treated with selumetinib showed inhibition of anchorage-independent growth, an indicator of in vivo tumorigenicity (P < 0.005), and decreases in the CD44+CD24−/low fraction, ALDH1 activity, and mammosphere-forming efficiency. Mice treated with selumetinib formed significantly fewer lung metastases than control mice injected with vehicle (P < 0.05). Our data demonstrate that MEK inhibitors can inhibit breast cancer stem cells and may have clinical potential for the prevention of metastasis in certain cases in which tumors are MAPK dependent. Mol Cancer Ther; 14(12); 2773–81. ©2015 AACR.

Bisphosphorylated PEA-15 Sensitizes Ovarian Cancer Cells to Paclitaxel by Impairing the Microtubule-Destabilizing Effect of SCLIP

Paclitaxel is a standard chemotherapeutic agent for ovarian cancer. PEA-15 (phosphoprotein enriched in astrocytes-15 kDa) regulates cell proliferation, autophagy, apoptosis, and glucose metabolism and also mediates AKT-dependent chemoresistance in breast cancer. The functions of PEA-15 are tightly regulated by its phosphorylation status at Ser104 and Ser116. However, the effect of PEA-15 phosphorylation status on chemosensitivity of cancer cells remains unknown. Here, we tested the hypothesis that PEA-15 phosphorylated at both Ser104 and Ser116 (pPEA-15) sensitizes ovarian cancer cells to paclitaxel. We first found that knockdown of PEA-15 in PEA-15–high expressing HEY and OVTOKO ovarian cancer cells resulted in paclitaxel resistance, whereas re-expression of PEA-15 in these cells led to paclitaxel sensitization. We next found that SKOV3.ip1-DD cells (expressing phosphomimetic PEA-15) were more sensitive to paclitaxel than SKOV3.ip1-AA cells (expressing nonphosphorylatable PEA-15). Compared with SKOV3.ip1-vector and SKOV3.ip1-AA cells, SKOV3.ip1-DD cells displayed reduced cell viability, inhibited anchorage-independent growth, and augmented apoptosis when treated with paclitaxel. Furthermore, HEY and OVTOKO cells displayed enhanced paclitaxel sensitivity when transiently overexpressing phosphomimetic PEA-15 and reduced paclitaxel sensitivity when transiently overexpressing nonphosphorylatable PEA-15. These results indicate that pPEA-15 sensitizes ovarian cancer cells to paclitaxel. cDNA microarray analysis suggested that SCLIP (SCG10-like protein), a microtubule-destabilizing protein, is involved in pPEA-15–mediated chemosensitization. We found that reduced expression and possibly posttranslational modification of SCLIP following paclitaxel treatment impaired the microtubule-destabilizing effect of SCLIP, thereby promoting induction of mitotic arrest and apoptosis by paclitaxel. Our findings highlight the importance of pPEA-15 as a promising target for improving the efficacy of paclitaxel-based therapy in ovarian cancer. Mol Cancer Ther; 12(6); 1099–111. ©2013 AACR.

Antitumor Activity of KW-2450 Against Triple-Negative Breast Cancer by Inhibiting Aurora A and B Kinases

Currently, no targeted drug is available for triple-negative breast cancer (TNBC), an aggressive breast cancer that does not express estrogen receptor, progesterone receptor, or HER2. TNBC has high mitotic activity, and, because Aurora A and B mitotic kinases drive cell division and are overexpressed in tumors with a high mitotic index, we hypothesized that inhibiting Aurora A and B produces a significant antitumor effect in TNBC. We tested this hypothesis by determining the antitumor effects of KW-2450, a multikinase inhibitor of both Aurora A and B kinases. We observed significant inhibitory activities of KW-2450 on cell viability, apoptosis, colony formation in agar, and mammosphere formation in TNBC cells. The growth of TNBC xenografts was significantly inhibited with KW-2450. In cell-cycle analysis, KW-2450 induced tetraploid accumulation followed by apoptosis or surviving octaploid (8N) cells, depending on dose. These phenotypes resembled those of Aurora B knockdown and complete pharmaceutical inhibition of Aurora A. We demonstrated that 8N cells resulting from KW-2450 treatment depended on the activation of mitogen-activated protein kinase kinase (MEK) for their survival. When treated with the MEK inhibitor selumetinib combined with KW-2450, compared with KW-2450 alone, the 8N cell population was significantly reduced and apoptosis was increased. Indeed, this combination showed synergistic antitumor effect in SUM149 TNBC xenografts. Collectively, Aurora A and B inhibition had a significant antitumor effect against TNBC, and this antitumor effect was maximized by the combination of selumetinib with Aurora A and B inhibition. Mol Cancer Ther; 14(12); 2687–99. ©2015 AACR.

Discovery of a potent inhibitor of MELK that inhibits expression of the anti-apoptotic protein Mcl-1 and TNBC cell growth

Despite recent advances in molecularly directed therapy, triple negative breast cancer (TNBC) remains one of the most aggressive forms of breast cancer, still without a suitable target for specific inhibitors. Maternal embryonic leucine zipper kinase (MELK) is highly expressed in TNBC, where level of overexpression correlates with poor prognosis and an aggressive disease course. Herein, we describe the discovery through targeted kinase inhibitor library screening, and structure-guided design of a series of ATP-competitive indolinone derivatives with subnanomolar inhibition constants towards MELK. The most potent compound, 17, inhibits the expression of the anti-apoptotic protein Mcl-1 and proliferation of TNBC cells exhibiting selectivity for cells expressing high levels of MELK. These studies suggest that further elaboration of 17 will furnish MELK-selective inhibitors with potential for development in preclinical models of TNBC and other cancers.

Serotonin Analogues as Inhibitors of Breast Cancer Cell Growth

Serotonin (5-hydroxytryptamine, 5-HT) is a critical local regulator of epithelial homeostasis in the breast and exerts its actions through a number of receptors. Dysregulation of serotonin signaling is reported to contribute to breast cancer pathophysiology by enhancing cell proliferation and promoting resistance to apoptosis. Preliminary analyses indicated that the potent 5-HT1B/1D serotonin receptor agonist 5-nonyloxytryptamine (5-NT), a triptan-like molecule, induced cell death in breast cancer cell lines. Thus, we synthesized a series of novel alkyloxytryptamine analogues, several of which decreased the viability of various human cancer cell lines. Proteomic and metabolomic analyses showed that compounds 6 and 10 induced apoptosis and interfered with signaling pathways that regulate protein translation and survival, such as the Akt/mTOR pathway, in triple-negative breast cancer cells.

Abstract 3774: KD06 is a novel anti-cancer drug that causes cell death in triple-negative breast cancer cell lines and tumor xenografts

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Development and screening of small molecule compounds for anti-cancer activity has been of prime interest to the scientific community following the success of targeted, large anti-cancer molecules such as therapeutic antibodies. Small molecules pass more easily through cell membranes and may cross the blood-brain barrier. KD06 is a small molecule triptan-like compound whose parent molecule binds and inhibits serotonin receptors. This compound increases apoptosis of the triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-157 via caspase activation. Treatment with KD06 also causes increased autophagy as well as activation of ER stress responses. The growth of tumor xenografts of MDA-MB-231 cells in nude mice are significantly inhibited by twice weekly treatment with 30mg/kg KD06. Analysis of signaling changes by KD06 using reverse phase protein array (RPPA) revealed significant decreases in Akt/mTOR signaling leading to decreased activation of the translation initiation factor 4E-BP1. Other notable changes included decreased expression of proteins important for mitosis such as Cyclin B1, Aurora B and PLK1, and increased phosphorylation of EGFR and increased expression of PDGFR. Analysis of PIP3 and ATP levels showed no change after treatment with KD06, indicating that decreased signaling through Akt/mTOR is not likely due to PI3K inhibition or AMPK activation. Immunofluorescence with KD06 treated cells revealed a change in cell shape after 4 hours of treatment that was reminiscent of cells treated with microtubule binding drugs. Akt localization was affected. These results imply that KD06 may have anti-cancer activity through its effect on microtubule dynamics, inhibiting proper localization and signaling of molecules important for survival and protein translation such as Akt and mTOR. Citation Format: Nancy D. Ebelt, Clint D J Taveres, Xuemei Xie, Youssef W. Naguib, Jiney Jose, Tinashe B. Ruwona, Ashwini K. Devkota, Jihyun Park, Tamer S. Kaoud, Eric V. Anslyn, Jeffrey T. Chang, Zhengrong Cui, Chandra Bartholomeusz, Kevin N. Dalby. KD06 is a novel anti-cancer drug that causes cell death in triple-negative breast cancer cell lines and tumor xenografts. [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 3774.

Abstract A17: TRPM7 kinase domain is involved in breast tumor cell metastasis

Abstracts: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX Introduction: TRPM7 (transient receptor potential melastatin 7) is a non-selective cation channel fused to protein kinase domain at the C-terminal whose activity is linked to the control of actomyosin contractility. TRPM7 mediates adhesion and migration of breast cancer cells and promotes breast tumor metastasis. The lack of cell-permeable inhibitors of the kinase domain represents a barrier to understand the kinase function. Methods: Herein, we discover the first small molecule (KD-1) that targets TRPM7 kinase activity and characterize its mechanism of action in-vitro, in-cells and in-vivo. By using this inhibitor, we are investigated the involvement of TRPM7 kinase domain in breast tumor cell metastasis. Results: Mg2+ starvation, which promotes TRPM7 kinase activity, induces phosphorylation of eEF2. Treatment of Mg2+-starved HEK293 cells with KD-1 decreased eEF2 phosphorylation, consistent with TRPM7 kinase activity suppression in-cells. KD-1 decreased the binding of Myosin IIB to TRPM7 in HEK293 and MDA-MB-231 cells. And when MDA-MB-231 and BT 549 cells were treated with increasing doses of KD-1, no change in cell viability was seen. Interestingly, KD-1 inhibited migration and invasion in the MDA-MB-231 and BT 549 cells that is reportedly regulated by TRPM7 kinase activity. Finally, the bioluminescent signals (to assess lung metastasis) were significantly lower in KD-1-treated mice (25 and 50 mg/kg/day) than in mice treated with vehicle control (P ≤0.05, 2-sided t- test.). Conclusion: The discovered compound represents the first inhibitor that targets the kinase activity of transient receptor potential melastatin 7 (TRPM7). Inhibition of TRPM7 kinase activity may reduce or block breast tumor progression and/or metastasis. Citation Format: Tamer S. Kaoud, Xuemei Xie, Jihyun Park, Clint D.J. Tavares, Shreya Mitra, Micael Cano, Mohamed F. Radwan, Chandra Bartholomeusz, Kevin N. Dalby. TRPM7 kinase domain is involved in breast tumor cell metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A17.

Abstract 1505: Suppression of triple-negative breast cancer tumorigenesis by targeting cancer stem cells through JNK/Notch1 signaling inhibition

Background: Triple-negative breast cancer (TNBC) is an aggressive disease with a poor prognosis and lacks targeted therapies. Basal-like TNBC exhibits hyperactivated JNK (c-Jun N-terminal kinase). JNK plays a vital role in malignant transformation of different cancers. However, it is unknown whether JNK signaling is a clinically relevant target in TNBC. Here, we tested the hypotheses that JNK signaling plays a fundamental role in TNBC pathogenesis by promoting self-renewal of cancer stem cells (CSCs). Methods: The role of JNK signaling in TNBC pathogenesis was determined by examining the effects of JNK signaling inhibition using siRNA or JNK-IN-8 (an ATP-competitive JNK inhibitor) and the effects of JNK overexpression on growth, migration, and invasion of TNBC cells using cell proliferation, migration, and invasion assays. CSC regulation by JNK signaling was examined using mammosphere-forming assay. The effect of JNK signaling inhibition on tumor growth was assessed using a xenograft mouse model. The downstream molecules involved in JNK signaling-mediated CSC regulation were identified using RT-PCR stem array analysis. Association between JNK and c-Jun was analyzed using a reverse phase protein array dataset of TNBC samples (n = 81). Disease-free survival (DFS) probability in TNBC patients was analyzed by the Kaplan-Meier method using a cDNA microarray dataset (n = 79). Results: Knockdown of JNK1 or JNK2 led to significant reductions in growth, migration, and invasion of TNBC cells, mammosphere formation, and ALDH1+ CSC subpopulation. Similar results were obtained when JNK signaling was inhibited using JNK-IN-8. In contrast, overexpression of JNK1 or JNK2 enhanced these cellular activities. Importantly, JNK signaling inhibition by JNK-IN-8 (25 mg/kg) reduced tumor growth by 60.88% (P Conclusion: JNK signaling regulates TNBC tumorigenesis by promoting CSC self-renewal via Notch1 signaling. JNK-IN-8 is a promising therapeutic agent for TNBC by targeting JNK/Notch1 signaling. Citation Format: Xuemei Xie, Tamer S. Kaoud, Ramakrishna Edupunganti, Tinghu Zhang, Takahiro Kogawa, Gaurav B. Chauhan, Dionysios N. Giannoukos, Yuan Qi, Debu Tripathy, Nathanael S. Gray, Kevin N. Dalby, Chandra Bartholomeusz, Naoto T. Ueno. Suppression of triple-negative breast cancer tumorigenesis by targeting cancer stem cells through JNK/Notch1 signaling inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1505. doi:10.1158/1538-7445.AM2015-1505

Abstract 750: JNK-IN-8: a novel covalent inhibitor targeting JNK signaling in triple-negative breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Patients with triple-negative breast cancer (TNBC) have a very poor prognosis due to metastasis and limited targeted therapies. Basal-like TNBC has hyperactivated JNK (c-Jun N-terminal kinase). JNK plays a vital role in malignant transformation in different cancers and self-renewal and maintenance of glioma cancer stem cells (CSCs). However, it is not known whether JNK signaling is a clinically relevant target in TNBC. We tested the hypotheses that JNK signaling plays a fundamental role in TNBC tumorigenesis and metastasis by promoting CSCs and that suppressing JNK signaling using JNK-IN-8 inhibits tumorigenicity and metastasis of TNBC cells. Methods: We examined the effects of JNK knockdown or overexpression on cellular functions of TNBC cells and CSC subpopulations in vitro. We also evaluated the anti-TNBC efficacy of JNK-IN-8, an ATP-directed covalent small molecule inhibitor of JNK that specifically binds the Cys116 on JNK. Results: In patients with TNBC (n=79), low c-Jun mRNA level was associated with better disease-free survival (DFS) (P=0.025), and there was a tendency (P=0.167) toward better DFS with low JNK1 mRNA level. No similar trend was observed for JNK2. These results suggest that JNK1 and c-Jun may have clinical significance in TNBC. Knockdown of JNK1 or JNK2 led to reductions in mammosphere formation, migration, and invasion in HCC70 and SUM149 cells. In contrast, overexpression of constitutively active MKK7-JNK1 or -JNK2 enhanced these cellular activities and increased the ALDH1+ subpopulation. These data indicate a potential role of JNK signaling in regulating TNBC tumorigenesis and metastasis, possibly by promoting CSCs. JNK-IN-8 suppressed proliferation, anchorage-independent growth, migration, and invasion in HCC70 and SUM149 cells. In addition, JNK-IN-8 also reduced mammosphere formation and the CD44+/CD24- and ALDH1+ subpopulations, suggesting that JNK-IN-8 can inhibit JNK signaling-mediated CSC self-renewal and maintenance. Furthermore, overexpression of mutant JNK1- or JNK2-C116S, in which Cys116 was replaced with Ser116, partially abolished the inhibitory effects of JNK-IN-8 on cellular functions, suggesting that JNK-IN-8 executes antitumor activity by specifically targeting JNK. Lastly, JNK inhibition by JNK-IN-8 led to down-regulation of Notch1, suggesting involvement of Notch1 in JNK signaling-mediated CSC regulation. Conclusion: JNK signaling regulates TNBC tumorigenesis and metastasis by promoting CSCs partly via Notch signaling. Our data demonstrate that JNK is an important therapeutic target in TNBC, providing a rationale for further preclinical and clinical development of a JNK-targeted therapy for TNBC. JNK-IN-8 is an effective inhibitor targeting JNK signaling in TNBC. Our studies justify searching for JNK isoform selective inhibitors since there is evidence that JNK1 and JNK2 can have opposing roles in certain settings. Citation Format: Xuemei Xie, Tamer S. Kaoud, Ramakrishna Edupunganti, Tinghu Zhang, Takahiro Kogawa, Gaurav B. Chauhan, Nathanael S. Gray, Chandra Bartholomeusz, Kevin N. Dalby, Naoto T. Ueno. JNK-IN-8: a novel covalent inhibitor targeting JNK signaling in triple-negative 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 750. doi:10.1158/1538-7445.AM2014-750

Abstract 3241: Preclinical development of JNK-targeted therapy for triple-negative breast cancer.

Patients with triple-negative breast cancer (TNBC), which is negative for estrogen receptor, progesterone receptor, and HER2, have a very poor prognosis due to metastasis and limited targeted therapies. JNK (c-Jun N-terminal kinase) signaling promotes tumorigenesis and metastasis and regulates cancer stem cell self-renewal and maintenance in glioma, and the JNK pathway is hyperactivated in basal-like TNBC. Therefore, we hypothesized that suppressing JNK activity inhibits tumorigenesis and metastasis of TNBC cells. To test our hypothesis, we generated several JNK-targeted JIP (JNK-interacting protein) mimetic peptides as well as the small molecule JNK inhibitor JNK-In-8 and assessed their therapeutic potential in TNBC cells. JIP mimetic peptides were designed and synthesized on the basis of the D-site of JIP, which is critical for JNK binding. All the peptides and JNK-In-8 specifically inhibited JNK kinase activity. Because of high JNK specificity, JIP2 (JIP peptide) and JNK-In-8 were chosen for bioactivity studies. JIP2 (10 μM) inhibited phosphorylation of JNK and its substrate c-Jun in HEK293 cells. Similar results were observed for JNK-In-8 (0.5 μM) in SUM149 and MDA-MB-231 cells. However, whereas JIP2 was not cytotoxic against SUM149 and MDA-MB-231 cells, JNK-In-8 (IC50=4.0 μM) significantly inhibited proliferation of these cells by inducing G1 cell-cycle arrest at low doses and apoptosis at high doses. Furthermore, SUM149 and MDA-MB-231 cells treated with JNK-In-8 (2.5 μM, P JIP2 (10 μM) strongly inhibited SUM149 cell migration (42.02%, P Interestingly, JNK-In-8 (2.5 μM) also reduced the proportion of CD44+ CD24- cells (SUM149: DMSO 50.23% vs JNK-In-8 40.41%) and that of ALDH1+ cells (SUM149: DMSO 38.25% vs JNK-In-8 25.84%; MDA-MB-231: DMSO 19.94% vs JNK-In-8 9.21%) as well as mammosphere formation (SUM149: 65.95%, P Our data demonstrate that JNK inhibitors may inhibit tumorigenesis and metastasis of TNBC cells by suppressing cancer stem cell maintenance and that JNK may serve as a promising target for TNBC therapy. Further studies will be conducted to determine the molecular mechanisms of JIP2- and JNK-In-8-mediated antitumor action in TNBC and the therapeutic efficacy of JIP2 and JNK-In-8 in a TNBC xenograft mouse model. Citation Format: Xuemei Xie, Tamer S. Kaoud, Ramakrishna Edupuganti, Rachel M. Sammons, Gabriel N. Hortobagyi, Naoto T. Ueno, Kevin N. Dalby, Chandra Bartholomeusz. Preclinical development of JNK-targeted therapy for triple-negative breast cancer. [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 3241. doi:10.1158/1538-7445.AM2013-3241