Michael S. Nakazawa

Dual CDK4/CDK6 Inhibition Induces Cell-Cycle Arrest and Senescence in Neuroblastoma

Purpose: Neuroblastoma is a pediatric cancer that continues to exact significant morbidity and mortality. Recently, a number of cell-cycle proteins, particularly those within the Cyclin D/CDK4/CDK6/RB network, have been shown to exert oncogenic roles in neuroblastoma, suggesting that their therapeutic exploitation might improve patient outcomes. Experimental Procedures: We evaluated the effect of dual CDK4/CDK6 inhibition on neuroblastoma viability using LEE011 (Novartis Oncology), a highly specific CDK4/6 inhibitor. Results: Treatment with LEE011 significantly reduced proliferation in 12 of 17 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations (mean IC50 = 307 ± 68 nmol/L in sensitive lines). LEE011 caused cell-cycle arrest and cellular senescence that was attributed to dose-dependent decreases in phosphorylated RB and FOXM1, respectively. In addition, responsiveness of neuroblastoma xenografts to LEE011 translated to the in vivo setting in that there was a direct correlation of in vitro IC50 values with degree of subcutaneous xenograft growth delay. Although our data indicate that neuroblastomas sensitive to LEE011 were more likely to contain genomic amplification of MYCN (P = 0.01), the identification of additional clinically accessible biomarkers is of high importance. Conclusions: Taken together, our data show that LEE011 is active in a large subset of neuroblastoma cell line and xenograft models, and supports the clinical development of this CDK4/6 inhibitor as a therapy for patients with this disease. Clin Cancer Res; 19(22); 6173–82. ©2013 AACR.

Hypoxia-Dependent Modification of Collagen Networks Promotes Sarcoma Metastasis

UNLABELLED Intratumoral hypoxia and expression of hypoxia-inducible factor-1α (HIF-1α) correlate with metastasis and poor survival in patients with sarcoma. We show here that hypoxia controls sarcoma metastasis through a novel mechanism wherein HIF-1α enhances expression of the intracellular enzyme procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2). We show that loss of HIF-1α or PLOD2 expression disrupts collagen modification, cell migration, and pulmonary metastasis (but not primary tumor growth) in allograft and autochthonous LSL-Kras(G12D/+); Trp53(fl/fl) murine sarcoma models. Furthermore, ectopic PLOD2 expression restores migration and metastatic potential in HIF-1α-deficient tumors, and analysis of human sarcomas reveals elevated HIF1A and PLOD2 expression in metastatic primary lesions. Pharmacologic inhibition of PLOD enzymatic activity suppresses metastases. Collectively, these data indicate that HIF-1α controls sarcoma metastasis through PLOD2-dependent collagen modification and organization in primary tumors. We conclude that PLOD2 is a novel therapeutic target in sarcomas and successful inhibition of this enzyme may reduce tumor cell dissemination. SIGNIFICANCE Undifferentiated pleomorphic sarcoma (UPS) is a commonly diagnosed and particularly aggressive sarcoma subtype in adults, which frequently and fatally metastasizes to the lung. Here, we show the potential use of a novel therapeutic target for the treatment of metastatic UPS, specifi cally the collagen-modifying enzyme PLOD2.

Oxygen availability and metabolic adaptations

Oxygen availability, along with the abundance of nutrients (such as glucose, glutamine, lipids and albumin), fluctuates significantly during tumour evolution and the recruitment of blood vessels, leukocytes and reactive fibroblasts to complex tumour microenvironments. As such, hypoxia and concomitant nutrient scarcity affect large gene expression programmes, signalling pathways, diverse metabolic reactions and various stress responses. This Review summarizes our current understanding of how these adaptations are integrated in hypoxic tumour cells and their role in disease progression.

Overcoming evasive resistance from vascular endothelial growth factor a inhibition in sarcomas by genetic or pharmacologic targeting of hypoxia-inducible factor 1α

Increased levels of hypoxia and hypoxia‐inducible factor 1α (HIF‐1α) in human sarcomas correlate with tumor progression and radiation resistance. Prolonged antiangiogenic therapy of tumors not only delays tumor growth but may also increase hypoxia and HIF‐1α activity. In our recent clinical trial, treatment with the vascular endothelial growth factor A (VEGF‐A) antibody, bevacizumab, followed by a combination of bevacizumab and radiation led to near complete necrosis in nearly half of sarcomas. Gene Set Enrichment Analysis of microarrays from pretreatment biopsies found that the Gene Ontology category “Response to hypoxia” was upregulated in poor responders and that the hierarchical clustering based on 140 hypoxia‐responsive genes reliably separated poor responders from good responders. The most commonly used chemotherapeutic drug for sarcomas, doxorubicin (Dox), was recently found to block HIF‐1α binding to DNA at low metronomic doses. In four sarcoma cell lines, HIF‐1α shRNA or Dox at low concentrations blocked HIF‐1α induction of VEGF‐A by 84–97% and carbonic anhydrase 9 by 83–93%. HT1080 sarcoma xenografts had increased hypoxia and/or HIF‐1α activity with increasing tumor size and with anti‐VEGF receptor antibody (DC101) treatment. Combining DC101 with HIF‐1α shRNA or metronomic Dox had a synergistic effect in suppressing growth of HT1080 xenografts, at least in part via induction of tumor endothelial cell apoptosis. In conclusion, sarcomas respond to increased hypoxia by expressing HIF‐1α target genes that may promote resistance to antiangiogenic and other therapies. HIF‐1α inhibition blocks this evasive resistance and augments destruction of the tumor vasculature.

Deregulation of the Hippo pathway in soft-tissue sarcoma promotes FOXM1 expression and tumorigenesis.

Significance Soft-tissue sarcomas are aggressive, often lethal tumors, which are understudied. Few therapies beyond standard resection and traditional chemotherapy/radiation are available. Sarcomas are diverse malignancies, including ∼65 distinct histological subtypes. The existence of common mechanisms underlying multiple subtypes has not previously been shown. We demonstrate that the Hippo pathway, an important regulator of cell proliferation, is deregulated in ≥25% of sarcomas, encompassing multiple commonly diagnosed subtypes. When control of the Hippo pathway is lost, expression of the effector protein Yes-Associated Protein (YAP) is stabilized, resulting in higher levels of proliferation. For the first time, to our knowledge, we show that YAP interacts with the forkhead box transcription factor FOXM1 to coregulate critical components of sarcomagenesis, specifically in fibrosarcoma, undifferentiated pleomorphic sarcomas, and liposarcomas. Genetic aberrations responsible for soft-tissue sarcoma formation in adults are largely unknown, with targeted therapies sorely needed for this complex and heterogeneous family of diseases. Here we report that that the Hippo pathway is deregulated in many soft-tissue sarcomas, resulting in elevated expression of the effector molecule Yes-Associated Protein (YAP). Based on data gathered from human sarcoma patients, a novel autochthonous mouse model, and mechanistic analyses, we determined that YAP-dependent expression of the transcription factor forkhead box M1 (FOXM1) is necessary for cell proliferation/tumorigenesis in a subset of soft-tissue sarcomas. Notably, FOXM1 directly interacts with the YAP transcriptional complex via TEAD1, resulting in coregulation of numerous critical pro-proliferation targets that enhance sarcoma progression. Finally, pharmacologic inhibition of FOXM1 decreases tumor size in vivo, making FOXM1 an attractive therapeutic target for the treatment of some sarcoma subtypes.

Epigenetic re-expression of HIF-2α suppresses soft tissue sarcoma growth.

In soft tissue sarcomas (STS), low intratumoural O2 (hypoxia) is a poor prognostic indicator. HIF-1α mediates key transcriptional responses to hypoxia, and promotes STS metastasis; however, the role of the related HIF-2α protein is unknown. Surprisingly, here we show that HIF-2α inhibits high-grade STS cell growth in vivo, as loss of HIF-2α promotes sarcoma proliferation and increases calcium and mTORC1 signalling in undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma. We find that most human STS have lower levels of EPAS1 (the gene encoding HIF-2α) expression relative to normal tissue. Many cancers, including STS, contain altered epigenetics, and our findings define an epigenetic mechanism whereby EPAS1 is silenced during sarcoma progression. The clinically approved HDAC inhibitor Vorinostat specifically increases HIF-2α, but not HIF-1α, accumulation in multiple STS subtypes. Vorinostat inhibits STS tumour growth, an effect ameliorated by HIF-2α deletion, implicating HIF-2α as a biomarker for Vorinostat efficacy in STS.

MYC Degradation under Low O2 Tension Promotes Survival by Evading Hypoxia-Induced Cell Death

ABSTRACT Cells encounter oxygen deprivation (hypoxia) in various physiological and pathological contexts. Adaptation to hypoxic stress occurs in part by suppressing MYC, a key regulator of cellular metabolism, proliferation, and survival. Hypoxia has been reported to inhibit MYC through multiple means, including disruption of MYC transcriptional complexes and decreased MYC protein abundance. Here we identify enhanced proteasomal degradation and cathepsin-mediated proteolysis as important mechanisms for hypoxic MYC inhibition in human colon carcinoma cells. MYC protein levels were similarly reduced in hypoxic primary keratinocytes. Increased MYC turnover at low O2 tension was dependent on the E3 ubiquitin ligases FBXW7 and DDB1, as well as hypoxic induction of cathepsins D and S. Reduced MYC protein levels coincided with hypoxic inhibition of RNA polymerase III-dependent MYC target genes, which MYC regulates independently of its binding partner MAX. Finally, MYC overexpression in hypoxic cells promoted cell cycle progression but also enhanced cell death via increased expression of the proapoptotic genes NOXA and PUMA. Collectively, these results indicate that hypoxic cells promote MYC degradation as an adaptive strategy to reduce proliferation, suppress biosynthetic processes, and promote cell survival under low O2 tension.

The aryl hydrocarbon receptor nuclear translocator is an essential regulator of murine hematopoietic stem cell viability.

Hypoxia-inducible factors (HIFs) are master regulators of the transcriptional response to low oxygen and play essential roles in embryonic development, tissue homeostasis, and disease. Recent studies have demonstrated that hematopoietic stem cells (HSCs) within the bone marrow localize to a hypoxic niche and that HIF-1α promotes HSC adaptation to stress. Because the related factor HIF-2α is also expressed in HSCs, the combined role of HIF-1α and HIF-2α in HSC maintenance is unclear. To this end, we have conditionally deleted the HIF-α dimerization partner, the aryl hydrocarbon receptor nuclear translocator (ARNT) in the hematopoietic system to ablate activity of both HIF-1α and HIF-2α and assessed the functional consequence of ARNT deficiency on fetal liver and adult hematopoiesis. We determined that ARNT is essential for adult and fetal HSC viability and homeostasis. Importantly, conditional knockout of both Hif-1α and Hif-2α phenocopied key aspects of these HSC phenotypes, demonstrating that the impact of Arnt deletion is primarily HIF dependent. ARNT-deficient long-term HSCs underwent apoptosis, potentially because of reduced B-cell lymphoma 2 (BCL-2) and vascular endothelial growth factor A (VEGF-A) expression. Our results suggest that HIF activity may regulate HSC homeostasis through these prosurvival factors.

Ischemia Induces Quiescence and Autophagy Dependence in Hepatocellular Carcinoma

Purpose To characterize hepatocellular carcinoma (HCC) cells surviving ischemia with respect to cell cycle kinetics, chemosensitivity, and molecular dependencies that may be exploited to potentiate treatment with transarterial embolization (TAE). Materials and Methods Animal studies were performed according to institutionally approved protocols. The growth kinetics of HCC cells were studied in standard and ischemic conditions. Viability and cell cycle kinetics were measured by using flow cytometry. Cytotoxicity profiling was performed by using a colorimetric cell proliferation assay. Analyses of the Cancer Genome Atlas HCC RNA-sequencing data were performed by using Ingenuity Pathway Analysis software. Activation of molecular mediators of autophagy was measured with Western blot analysis and fluorescence microscopy. In vivo TAE was performed in a rat model of HCC with (n = 5) and without (n = 5) the autophagy inhibitor Lys05. Statistical analyses were performed by using GraphPad software. Results HCC cells survived ischemia with an up to 43% increase in the fraction of quiescent cells as compared with cells grown in standard conditions (P < .004). Neither doxorubicin nor mitomycin C potentiated the cytotoxic effects of ischemia. Gene-set analysis revealed an increase in mRNA expression of the mediators of autophagy (eg, CDKN2A, PPP2R2C, and TRAF2) in HCC as compared with normal liver. Cells surviving ischemia were autophagy dependent. Combination therapy coupling autophagy inhibition and TAE in a rat model of HCC resulted in a 21% increase in tumor necrosis compared with TAE alone (P = .044). Conclusion Ischemia induces quiescence in surviving HCC cells, resulting in a dependence on autophagy, providing a potential therapeutic target for combination therapy with TAE.

Abstract 960: Deregulation of the Hippo pathway in soft tissue sarcoma promotes FOXM1 expression and tumorigenesis

The genetic aberrations responsible for soft tissue sarcoma formation in adults are largely unknown, and targeted therapies are sorely needed for this complex and heterogeneous family of diseases. Here we report that that the Hippo pathway is deregulated in many soft tissue sarcomas, resulting in elevated expression of the effector molecule YAP. Based on data gathered from human sarcoma patients, we concluded that more than 40% of human sarcomas have lost one or more copies of upstream HIPPO pathway regulators including SAV1, LATS2, and NF2. Loss of these molecules deactivates the HIPPO pathway, permitting uncontrolled proliferation and growth. Expression of active YAP is the direct result of this copy number loss and we have found YAP to be elevated in primary human fibrosarscomas, liposarcomas, and pleomorphic tumors. Pharmacologic inhibition of YAP significantly impaired primary sarcomagenesis in a murine allograft model. Using a novel autochthonous mouse model and mechanistic analyses in human and murine sarcoma cells, we determined that YAP-dependent expression of the transcription factor, FOXM1, is necessary for sarcoma cell proliferation and tumorigenesis. FOXM1 expression is elevated in multiple human soft tissue sarcoma subtypes. Notably, FOXM1 directly interacts with the YAP transcriptional complex via TEAD1, resulting in co-regulation of numerous critical pro-proliferation targets that enhance sarcoma progression. Finally, pharmacologic inhibition of FOXM1 decreases tumor size in vivo, making FOXM1 an attractive therapeutic target for the treatment of sarcomas. Citation Format: T.S. Karin Eisinger, Vera Mucaj, Kevin Biju, Michael Nakazawa, Mercy Gohil, Timothy Cash, Sam Yoon, Nicolas Skuli, Kyung Min park, Sharon Gerecht, Celeste Simon. Deregulation of the Hippo pathway in soft tissue sarcoma promotes FOXM1 expression and tumorigenesis. [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 960. doi:10.1158/1538-7445.AM2015-960