Fengjuan Fan

Targeting the Tumor Microenvironment: Focus on Angiogenesis

Tumorigenesis is a complex multistep process involving not only genetic and epigenetic changes in the tumor cell but also selective supportive conditions of the deregulated tumor microenvironment. One key compartment of the microenvironment is the vascular niche. The role of angiogenesis in solid tumors but also in hematologic malignancies is now well established. Research on angiogenesis in general, and vascular endothelial growth factor in particular, is a major focus in biomedicine and has led to the clinical approval of several antiangiogenic agents including thalidomide, bevacizumab, sorafenib, sunitinib, pazopanib, temesirolimus, and everolimus. Indeed, antiangiogenic agents have significantly changed treatment strategies in solid tumors (colorectal cancer, renal cell carcinoma, and breast cancer) and multiple myeloma. Here we illustrate important aspects in the interrelationship between tumor cells and the microenvironment leading to tumor progression, with focus on angiogenesis, and summarize derived targeted therapies.

Mcl-1 confers protection of Her2-positive breast cancer cells to hypoxia: therapeutic implications

BackgroundMolecular mechanisms leading to the adaptation of breast cancer (BC) cells to hypoxia are largely unknown. The anti-apoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1) is frequently amplified in BC; and elevated Mcl-1 levels have been correlated with poor prognosis. Here we investigated the pathophysiologic role of Mcl-1 in Her2-positive BC cells under hypoxic conditions.MethodsRNA interference and a novel small molecule inhibitor, EU-5346, were used to examine the role of Mcl-1 in Her2-positive BC cell lines and primary BC cells (sensitive or intrinsically resistant to Her2 inhibitors) under hypoxic conditions (using a hypoxic incubation chamber). Mechanisms-of-action were investigated by RT-PCR, mitochondrial isolation, as well as immunoprecipitation/blotting analysis, and microscopy. The specificity against Mcl-1 of the novel small molecule inhibitor EU5346 was verified in Mcl-1Δ/nullversus Mcl-1wt/wt Murine Embryonic Fibroblasts (MEFs). Proliferation, survival, and spheroid formation were assessed in response to Mcl-1 and Her2 inhibition.ResultsWe demonstrate for a strong correlation between high Mcl-1 protein levels and hypoxia, predominantly in Her2-positive BC cells. Surprisingly, genetic depletion of Mcl-1 decreased Her2 and Hif-1α levels followed by inhibition of BC cell survival. In contrast, Mcl-1 protein levels were not downregulated after genetic depletion of Her2 indicating a regulatory role of Mcl-1 upstream of Her2. Indeed, Mcl-1 and Her2 co-localize within the mitochondrial fraction and form a Mcl-1/Her2- protein complex. Similar to genetically targeting Mcl-1 the novel small molecule Mcl-1 inhibitor EU-5346 induced cell death and decreased spheroid formation in Her2-positive BC cells. Of interest, EU-5346 induced ubiquitination of Mcl-1- bound Her2 demonstrating a previously unknown role for Mcl-1 to stabilize Her2 protein levels. Importantly, targeting Mcl-1 was also active in Her2-positive BC cells resistant to Her2 inhibitors, including a brain-primed Her2-positive cell line.ConclusionOur data demonstrate a critical role of Mcl-1 in Her2-positive BC cell survival under hypoxic conditions and provide the preclinical framework for the therapeutic use of novel Mcl-1- targeting agents to improve patient outcome in BC.

Pre-Osteoblasts Stimulate Migration of Breast Cancer Cells via the HGF/MET Pathway.

INTRODUCTION The occurrence of skeletal metastases in cancer, e.g. breast cancer (BC), deteriorates patient life expectancy and quality-of-life. Current treatment options against tumor-associated bone disease are limited to anti-resorptive therapies and aimed towards palliation. There remains a lack of therapeutic approaches, which reverse or even prevent the development of bone metastases. Recent studies demonstrate that not only osteoclasts (OCs), but also osteoblasts (OBs) play a central role in the pathogenesis of skeletal metastases, partly by producing hepatocyte growth factor (HGF), which promotes tumor cell migration and seeding into the bone. OBs consist of a heterogeneous cell pool with respect to their maturation stage and function. Recent studies highlight the critical role of pre-OBs in hematopoiesis. Whether the development of bone metastases can be attributed to a particular OB maturation stage is currently unknown. METHODS AND RESULTS Pre-OBs were generated from healthy donor (HD)-derived bone marrow stromal cells (BMSC) as well as the BMSC line KM105 and defined as ALPlow OPNlow RUNX2high OSX high CD166high. Conditioned media (CM) of pre-OBs, but not of undifferentiated cells or mature OBs, enhanced migration of metastatic BC cells. Importantly, HGF mRNA was significantly up-regulated in pre-OBs versus mature OBs, and CM of pre-OBs activated the MET signaling pathway. Highlighting a key role for HGF, CM from HGF-negative pre-OBs derived from the BMSC line HS27A did not support migration of BC cells. Genetically (siMET) or pharmacologically (INCB28060) targeting MET inhibited both HGF- and pre-OB CM- mediated BC cell migration. CONCLUSIONS Our data demonstrate for the first time a role for pre-OBs in mediating HGF/MET- dependent migration of BC cells and strongly support the clinical evaluation of INCB28060 and other MET inhibitors to limit and/or prevent BC-associated bone metastases.

Abstract 2912: The AP-1 transcription factor JunB promotes multiple myeloma cell proliferation, survival and drug resistance in the bone marrow microenvironment

Introduction: The activator protein-1 (AP-1) transcription factor has been implicated in a multitude of physiologic processes, but also tumorigenesis. In multiple myeloma (MM), the role of AP-1 is largely unknown. Experimental procedures: MM cell lines and primary tumor cells were co-cultured with primary bone marrow stromal cells (BMSCs) or BMSC lines. AP-1 expression was measured by western blot analysis and real-time PCR. AP-1 activation was determined using TransAM AP-1 assay kit. To investigate the upstream regulators of JunB, cytokine array and specific inhibitors were used followed by 3H-thymidine incorporation, western blot and TransAM AP-1 assays. To delineate the specific functional role of JunB in MM pathogenesis, we used pLKO.1- JunB shRNA (shJunB) and pLKO.1- scrambled shRNA (SCR) vectors for constitutive knockdown, as well as pMSCV-JunB-ER-IRES-GFP and empty vectors for inducible overexpression, together with 3H-thymidine incorporation, alamarBlue, flow cytometry and western blot analysis, as well as gene expression profiling (GEP). To evaluate the functional role of JunB in vivo, a MM xenograft mouse model was used. Results: Co-cultures of MM cells with BMSCs rapidly and strongly induced sustained expression and activation of JunB, but not of other AP-1 family members. Induction of JunB was predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Indeed, IL-6 stimulation of MM.1S cells resulted in rapid and strong upregulation of JunB. Conversely, anti-IL-6 receptor antibody tocilizumab blocked BMSC-induced JunB expression and activation. Pharmacologic inhibition identified the requirement of the MEK/ERK and NF-κB pathways for BMSC-induced JunB expression. Functionally, significant inhibition of proliferation was observed in MM cells carrying pLKO.1- shJunB, but not pLKO.1-SCR. Importantly, knockdown of other AP-1 family members had minor effects on MM cell proliferation. Moreover, GEP performed on MM.1S- shJunB cells co-cultured with BMSCs as well as data analysis of a patient cohort using Gene Set Enrichment Analysis (GSEA) suggested a key role for JunB in the regulation of Mcl-1 and c-Myc expression. Furthermore, knockdown of JunB overcame resistance of MM cells to dexamethasone. Conversely, 4-OHT treatment of MM cell lines transduced with JunB-ER but not control vector induced significant JunB/AP-1 luciferase activity and protected MM cells against bortezomib-induced apoptosis and ER stress. Confirming our in vitro data, preliminary results show significant inhibition of tumor growth in a xenograft mouse model inoculated with inducible Tet-shJunB-GFP but not Tet-SCR-GFP MM.1S cells upon treatment with doxycycline. Conclusion: Taken together, our data demonstrate for the first time an important and surprising role of JunB/AP-1 in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM. Citation Format: Fengjuan Fan, Sonia Vallet, Muhammad Hasan Bashari, Mostafa Jarahian, Eugenio Morelli, Dirk Hose, Latifa Bakiri, Claudia Ball, Hanno Glimm, Martin Sattler, Hartmut Goldschmidt, Giovanni Tonon, Pierfrancesco Tassone, Erwin F. Wagner, Dirk Jager, Klaus Podar. The AP-1 transcription factor JunB promotes multiple myeloma cell proliferation, survival and drug resistance in the bone marrow microenvironment. [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 2912.

Abstract A60: Mcl-1 confers protection of Her2-positive breast cancer cells to hypoxia: Therapeutic implications

Molecular mechanisms leading to the adaptation of breast cancer (BC) cells to hypoxia are largely unknown. The anti-apoptotic Bcl-2 family member Myeloid cell leukemia-1 (Mcl-1) is frequently amplified in BC; and elevated Mcl-1 levels have been correlated with poor prognosis. Here, we demonstrate for the first time strong correlation between Mcl-1 protein levels and hypoxia, predominantly in Her2-positive BC cells. Surprisingly, genetic depletion of Mcl-1 decreased Her2 and Hif-1α levels followed by inhibition of BC cell survival. In contrast, Mcl-1 protein levels were not downregulated after genetic depletion of Her2 indicating a regulatory role of Mcl-1 upstream of Her2. Indeed, Mcl-1 and Her2 co-localize within the mitochondrial fraction and form a Mcl-1/Her2- protein complex. Similar to genetically targeting Mcl-1 the novel small molecule Mcl-1 inhibitor EU-5346 induced cell death and decreased spheroid formation in Her2-positive BC cells. Of interest, EU-5346 induced ubiquitination of Mcl-1- bound Her2 demonstrating a previously unknown role for Mcl-1 to stabilize Her2 protein levels. Importantly, targeting Mcl-1 was also active in Her2-positive BC cells resistant to Her2 inhibitors, including a brain-primed Her2-positive cell line. In summary, our data demonstrate a critical role of Mcl-1 in Her2-positive BC cell survival under hypoxic conditions and provide the preclinical framework for the therapeutic use of novel Mcl-1- targeting agents to improve patient outcome in BC. Citation Format: Muhammad Hasan Bashari, Fengjuan Fan, Sonia Vallet, M. Arn, Michael H. Cardone, Philipp Beckhove, Andreas Schneeweiss, Martin Sattler, Joseph T. Opferman, Dirk Jager, Klaus Podar. Mcl-1 confers protection of Her2-positive breast cancer cells to hypoxia: Therapeutic implications. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A60.

Abstract 3383: JunB/AP-1 controls MM cell proliferation, survival and drug resistance in the bone marrow microenvironment

The activator protein-1 (AP-1) transcription factor is a dimeric complex that comprises members of the JUN, FOS, activating transcription factor (ATF) and musculoaponeurotic fibrosarcoma (MAF) protein families. Resultant heterodimers and homodimers determine the genes that are regulated by AP-1. Functionally, AP-1 has been implicated in a multitude of biological processes but also tumorigenesis, dependent on the cell type and the pathophysiologic context. In multiple myeloma (MM), the role of AP-1 is largely unknown. The present study focuses on the impact of JunB, a central member of the AP-1 family, in MM growth, survival and drug resistance. First, our data demonstrate rapid and strong induction of AP-1/JunB expression in MM cell lines and primary tumor cells upon co-culture with bone marrow stromal cells (BMSCs), i.e. isotypic primary BMSCs as well as BMSC lines KM-105 and HS-27A. Importantly, utilizing a non-contact co-culture system, we show that this effect is predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Consistent with gene expression profiling data, JunB mRNA levels detected by quantitative real-time PCR are increased in MM cells when co-cultured with BMSCs. Specifically, we found that upregulation of JunB occurs predominantly at the translational level, since cycloheximide, but not actinomycin D or MG-132 significantly blocked BMSC- induced JunB expression. Pharmacologic inhibition was used next in order to identify upstream signaling pathways, which mediate BMSC- induced JunB upregulation in MM cells. Our data demonstrate that activation of MEK/MAPK or NF-kB, but not PI3K/Akt is required for induction of JunB expression and AP-1 transcriptional activity. To delineate the specific functional role of JunB in MM pathogenesis, we subsequently generated stable MM cell lines carrying the pLKO.1-puro-JunB shRNA vector or the pLKO.1-puro control vector, respectively. Our preliminary data show significantly decreased proliferation and resistance to chemotherapy in MM/ pLKO.1-puro-JunB shRNA but not MM/ pLKO.1-puro control cells when co-cultured with BMSCs. Consistently, 4-hydroxytamoxifen (4-HT) treatment of MM cells stably transduced with pBabe-puro-JunB-ER-IRES-GFP but not of MM cells transduced with pBabe-puro-IRES-GFP promotes tumor cell proliferation, survival and drug resistance, independent of BMSC co-culture. Ongoing studies aim to identify downstream effectors of JunB, using gene array profiling of MM/ pLKO.1-puro-JunB shRNA cells versus MM/ pLKO.1-puro control cells. Furthermore, we are conducting a survey of published and unpublished datasets to uncover the clinical and prognostic relevance of JunB expression in MM patient samples. In summary, our data demonstrate for the first time an important role of AP-1/JunB in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM. Citation Format: Fengjuan Fan, Sonia Vallet, Martin Sattler, Giovanni Tonon, Muhammad Hasan Bashari, Latifa Bakiri, Hartmut Goldschmidt, Erwin F. Wagner, Dirk Jaeger, Klaus Podar. JunB/AP-1 controls MM cell proliferation, survival and drug resistance in the bone marrow microenvironment. [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 3383. doi:10.1158/1538-7445.AM2014-3383

Abstract 1734: Drug- induced generation of Mcl-1128-350 fragment triggers apoptosis in multiple myeloma cells through upregulation of c-Jun.

Myeloid cell leukemia-1 (Mcl-1) gene, a pro-survival member of the Bcl-2 family, has been suggested to play a key role in Multiple Myeloma (MM) pathogenesis and drug resistance. Our present data demonstrate significant overexpression of Mcl-1 gene in a data set comparing samples from more than 500 MM patients with samples from healthy donors. These findings indicate Mcl-1 as a potential therapeutic target in MM. One strategy to inhibit Mcl-1 activity in MM and other malignancies is the induction of proteasome- independent Mcl-1 degradation. Indeed, our own and other previous studies demonstrate that a caspase- induced 28 kD Mcl-1 fragment, Mcl-1 128-350 inhibits MM cell proliferation and survival. Importantly, our present results show that bortezomib, staurosporine and adaphostin, but not doxorubicin, thalidomide or enzastaurin induce generation of Mcl-1 128-350 in MM cells. We next sought to identify molecular sequelae downstream of Mcl-1 128-350 , which mediate its pro-apoptotic effect. Surprisingly, we observed nuclear accumulation of drug- induced or transfected Mcl-1 128-35 0 , followed by elevated mRNA and protein levels of c-Jun as well as enhanced AP-1 reporter activity. Supporting a key role of Mcl-1 128-350 in c-Jun transcription, drug- induced c-Jun upregulation was blocked after introduction of point mutations into the two highly conserved Mcl-1 caspase- cleavage sites Asp127 and Asp157. Consistent with these data, treatment with bortezomib triggered c-Jun upregulation followed by apoptosis in Mcl-1wt/wt, but not Mcl-1d/null murine embryonic fibroblasts (MEFs). Conversely, transfection of a plasmid carrying Mcl-1wt into Mcl-1d/null MEFs restored bortezomib- induced Mcl-1 fragementation, c-Jun upregulation and cell apoptosis. Importantly, Mcl-1 sequencing in several MM cell lines and primary cells didn9t show the existence of point mutations in Mcl-1 caspase- cleavage sites, excluding a potential mechanism of drug resistance. In order to identify c-Jun downstream effectors of MM cell apoptosis, gene array profiling was finally performed on MM cells transfected with Mcl-1wt or Mcl-1 128-350 . Taken together, the present study demonstrates a promising therapeutic role of Mcl-1 128-350 in MM. Moreover, our preliminary data indicate that a similar therapeutic role of Mcl-1 128-350 may also exist in a variety of solid and other hematologic malignancies. Citation Format: Fengjuan Fan, Giovanni Tonon, Martin Sattler, Hartmut Goldschmidt, Joseph Opferman, Kenneth C. Anderson, Dirk Jager, Klaus Podar. Drug- induced generation of Mcl-1 128-350 fragment triggers apoptosis in multiple myeloma cells through upregulation of c-Jun. [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 1734. doi:10.1158/1538-7445.AM2013-1734