Abstract: Our studies have revealed an unrecognized mechanism for regulating the activity of p53 during the cell cycle. We show, using multiple approaches, that p53 is degraded as a consequence of its phosphorylation by Aurora B. This degradation ultimately prevents p53 from performing its role as a guardian of the genome. Taken together with our earlier study on the effects of the Aurora B kinase inhibitor AZD1152 on breast cancer cells and preclinical evaluations of this drug for treating diverse human tumors, we conclude that Aurora B inhibitors may prove to be effective in treating cancer cells expressing functional p53 molecules. Also, we previously showed that specific inhibition of Aurora B expression is sufficient to inhibit tumor growth and induce the regression of tumors. Significantly, we here show that a specific inhibitor (AZD1152) of Aurora B can cause p53 elevation, thereby increasing p53 target gene expression in a cancer xenograft model. As a result, inhibitor of Aurora B can reduce cell survival through increasing the p53 up-regulated mediator of apoptosis p53 up-regulated modulator of apoptosis (PUMA) and cause cell cycle inhibition through inducing CDK inhibitor p21. These data are consistent with biochemical studies in cell lines. Our present study defines the molecular mechanisms involved in inhibition of tumor growth by an Aurora B inhibitor. General suppression of transcription is observed during mitosis ( 4 ); therefore, Aurora B-mediated p53 transcriptional suppression will not play a role during mitosis. The work by Cross et al. ( 2 ) shows that p53 is involved in facilitating chromosome segregation to ensure the maintenance of diploid cells. Aurora B may coordinate with p53 to mediate the spindle checkpoint ( 2 ) and aid progression through mitosis. Given that Aurora B deregulation also results in polyploidy, the interplay between p53 and Aurora B is conceivably important for spindle checkpoint. The functional significance of Aurora B–p53 interaction during different stages of mitosis remains to be investigated, but p53 is involved in spindle checkpoint ( 2 ); our data serve to confirm its presence in the spindle checkpoint machinery. It is important to point out that the binding between Aurora B and p53 decreases after the end of mitosis because of the down-regulation of Aurora B by E3 ubiquitin ligase anaphase promoting complex. After mitosis, Aurora B is recovering from degradation and again binding p53, and thus, it potentially prevents p53 from arresting the cell cycle at G1 by maintaining a negative impact on p53. The enhanced degradation of phosphorylated p53 leads to the finding that its transcriptional activity on cell cycle control gene was diminished (such as p21 CDK inhibitor) when Aurora B was overexpressed. Moreover, we were able to show that p53-mediated gene transcription was enhanced when Aurora B expression was inhibited. These data provide a rationale for the role of Aurora B in interphase, which is to antagonize the inhibition of cell cycle progression by p53 and allow entry of cells into mitosis. To answer this question, we used bimolecular fluorescence complementation, which allows direct observation of the interaction between two proteins in living cells, and showed that Aurora B and p53 interact during interphase and mitosis ( Fig. P1 ). Immunofluorescence studies showed that, during mitosis, p53 associates with Aurora B located at centromeres at prometaphase or the middle zone of the cleavage furrow at the anaphase–telophase border. We were also able to show that Survivin, a protein component of CPC localized at the centromeres to activate Aurora B ( 3 ), colocalized with Aurora B and p53 to the DNA of prometaphase cells. We also showed that Aurora B and p53 could be coimmunoprecipitated with specific antibodies. Using the technique, we were also able to show the Aurora B–p53 association in every phase of the cell cycle except late M phase, when Aurora B is known to be degraded. The interaction in interphase is quite surprising, because Aurora B has been shown to function exclusively during mitosis. We then showed that Aurora B phosphorylates p53 at three predicted Aurora B sites (S183, T211, and S215). We also showed that these phosphorylations lead to enhanced degradation of p53 through ubiquitination, which is mediated by the murine double minute 2 (MDM2) regulatory protein, an E3 ubiquitin ligase of p53. Moreover, AZD1152-hydroxyquinazoline pyrazole anilide (HQPA), which specifically inhibits the kinase activity of Aurora B, was able to block p53 ubiquitination in a dose-dependent manner. These data provide insights into the contribution of Aurora B-mediated p53 phosphorylation in regulating p53 stability. The regulation of mitosis ensures the equal segregation of chromosomes to daughter cells, and Aurora B plays a critical role in this process as a component of the chromosomal passenger protein complex (CPC). CPC is located on the chromosome arms during prophase and at the centromeres during prometaphase and metaphase ( 1 ). Aurora B subsequently localizes to the midbody during cytokinesis and participates in ensuring the correct attachment of chromosomes to spindle microtubules (spindle checkpoint) and equal distribution of chromosomes. The effects of Aurora B are exerted by its phosphorylation on specific proteins of the mitotic apparatus. The p53 protein is also involved in facilitating chromosome segregation to ensure the maintenance of diploid cells because cells deficient in p53 expression become tetraploid ( 2 ). Thus, it raises the question of whether Aurora B and p53 are functionally related. Aurora B, a protein kinase, plays a key role in mitosis by maintaining correct chromosome segregation and progression of cells through mitosis. Cancer cells frequently express Aurora B at unusually high levels, leading to dysregulated mitosis and therefore, causing unequal chromosome segregation, which may confer a growth advantage. The tumor suppressor protein p53 guards the genome by delaying or arresting the cell cycle at specific checkpoints (G1/S) when DNA is damaged and prevents damaged cells from entering mitosis (G2/M checkpoint). Despite intensive studies on p53 for more than three decades, the exact mechanism by which it regulates mitotic checkpoint is unknown. Whether Aurora B and p53 are coordinately regulated during the cell cycle remains to be determined, and there is no report to our knowledge suggesting that Aurora B functions in processes other than mitosis. By studying cells synchronized to divide at the same time, we show here that Aurora B and p53 interact during all stages of the cell cycle except during late M phase, when Aurora B is degraded. Moreover, we show that Aurora B is a negative regulator of p53 and that, when overexpressed, it affects the ability of p53 to mitigate the detrimental consequences of DNA damage and control the mitotic checkpoint.

Abstract: Our recent study found that activation of signal transducer and activator of transcription 3 (Stat3) is up-regulated in human brain metastatic cells and contributes to brain metastasis of melanoma. However, the molecular mechanisms underlying this increased Stat3 activation and effect on brain metastasis are unknown. In this report, we showed that the expression of Janus-activated kinase 2 (JAK2), a Stat3 activator, was increased, whereas the expression of a negative regulator of Stat3, suppressor of cytokine signaling-1 (SOCS-1), was reduced in the brain metastatic melanoma cell line A375Br, relative to that in the parental A375P cell line. Consistently, SOCS-1 expression was also lower in the human brain metastatic tissues than in the primary melanoma tissues. Mechanistically, increased JAK2 expression in the A375Br cells was due to, at least in part, its decreased degradation, which was directly correlated with low expression of SOCS-1. Moreover, restoration of SOCS-1 expression resulted in the inhibition of Stat3 activation, whereas depletion of SOCS-1 up-regulated Stat3 activation. These clinical, experimental, and mechanistic findings strongly suggest that increased activation of Stat3 in brain metastatic melanoma cells might be due to decreased SOCS-1 expression. Furthermore, restoration of SOCS-1 expression in brain metastatic A375Br cells significantly inhibited brain metastasis in animal models (P & lt; 0.001). Additionally, alterations of SOCS-1 expression profoundly affected the expression of matrix metalloproteinase-2 (MMP-2), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) and the melanoma cell invasion and angiogenesis. Collectively, these data suggest that the loss of SOCS-1 expression is a critical event, leading to elevated Stat3 signaling and overexpression of MMP-2, bFGF, and VEGF, as well as enhanced invasion and angiogenesis of melanoma cells, consequently promoting brain metastasis. [Cancer Res 2008;68(23):9634–42]

Abstract: Tumor-infiltrating myeloid cells are the most abundant leukocyte population within tumors. Molecular cues from the tumor microenvironment promote the differentiation of immature myeloid cells toward an immunosuppressive phenotype. However, the in situ dynamics of the transcriptional reprogramming underlying this process are poorly understood. Therefore, we applied single cell RNA-seq (scRNAseq) to computationally investigate the cellular composition and transcriptional dynamics of tumor and adjacent normal tissues from 4 early-stage non-small cell lung cancer (NSCLC) patients. Our scRNA-seq analyses identified 11,485 cells that varied in identity and gene expression traits between normal and tumor tissues. Among these, myeloid cell populations exhibited the most diverse changes between tumor and normal tissues, consistent with tumor-mediated reprogramming. Through trajectory analysis, we identified a differentiation path from CD14+ monocytes to M2 macrophages (monocyte-to-M2). This differentiation path was reproducible across patients, accompanied by increased expression of genes (e.g. MRC1/CD206, MSR1/CD204, PPARG, TREM2) with significantly enriched functions (Oxidative phosphorylation and P53 pathway) and decreased expression of genes (e.g. CXCL2, IL1B) with significantly enriched functions (TNFa signaling via NF-kB and inflammatory response). Our analysis further identified a co-regulatory network implicating upstream transcription factors (JUN, NFKBIA) in monocyte-to-M2 differentiation, and activated ligand-receptor interactions (e.g. SFTPA1-TLR2, ICAM1-ITGAM) suggesting intratumoral mechanisms whereby epithelial cells stimulate monocyte-to-M2 differentiation. Overall, our analysis identified the prevalent monocyte-to-M2 differentiation in NSCLC, accompanied by an intricate transcriptional reprogramming mediated by specific transcriptional activators and intercellular crosstalk involving ligand-receptor interactions. Citation Format: Qianqian Song, Gregory A. Hawkins, Leonard Wudel, Ping-Chieh Chou, Elizabeth Forbes, Ashok K. Pullikuth, Liang Liu, Guangxu Jin, Lou Craddock, Umit Topaloglu, Gregory Kucera, Stacey O’Neill, Edward A. Levine, Peiqing Sun, Kounosuke Watabe, Yong Lu, Martha A. Alexander-Miller, Boris Pasche, Lance D. Miller, Wei Zhang. Dissecting intratumoral cell-cell interactions in myeloid reprogramming by single cell RNA-seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3391.

Abstract: Hyperpolarized [1-13C]-pyruvate has shown tremendous promise as an agent for imaging tumor metabolism with unprecedented sensitivity and specificity. Imaging hyperpolarized substrates by magnetic resonance is unlike traditional MRI because signals are highly transient and their spatial distribution varies continuously over their observable lifetime. Therefore, new imaging approaches are needed to ensure optimal measurement under these circumstances. Constrained reconstruction algorithms can integrate prior information, including biophysical models of the substrate/target interaction, to reduce the amount of data that is required for image analysis and reconstruction. In this study, we show that metabolic MRI with hyperpolarized pyruvate is biased by tumor perfusion and present a new pharmacokinetic model for hyperpolarized substrates that accounts for these effects. The suitability of this model is confirmed by statistical comparison with alternates using data from 55 dynamic spectroscopic measurements in normal animals and murine models of anaplastic thyroid cancer, glioblastoma, and triple-negative breast cancer. The kinetic model was then integrated into a constrained reconstruction algorithm and feasibility was tested using significantly undersampled imaging data from tumor-bearing animals. Compared with naïve image reconstruction, this approach requires far fewer signal-depleting excitations and focuses analysis and reconstruction on new information that is uniquely available from hyperpolarized pyruvate and its metabolites, thus improving the reproducibility and accuracy of metabolic imaging measurements. Cancer Res; 75(22); 4708–17. ©2015 AACR.

Abstract: Glioblastoma is one of the most lethal malignancies with the poorest prognosis among tumors of the central nervous system. Fusion genes are common chromosomal aberrations in many cancers that may give rise to an in-frame fusion protein with oncogenic function. These fusion genes can be used as prognostic markers as well as drug targets in clinical practice. The FGFR3-TACC3 (F3T3) fusion gene was discovered in glioblastoma, with an occurrence rate of up to 8.3% and leading to uncontrolled proliferation and chromosomal instability. Our studies have shown that glioblastoma cells harboring the F3T3 are resistant to the frontline glioblastoma drug Temozolomide (TMZ). Using the Comet assay, we show that TMZ-mediated DNA damage is repaired more rapidly in cells harboring the F3T3 compared to control. Our studies on the mechanism of this drug resistance show that the F3T3 confers an aberrant activation of FGFR and ERK pathways when treated with TMZ. To further explore druggable target(s) to overcome resistance caused by F3T3, we immunoprecipitated (IP) the proteins that interact with F3T3 in U251 glioblastoma cells overexpressing the F3T3 or wildtype FGFR3, followed by LC-MS/MS analyses. Our quantitative proteomic analysis revealed interactions of the F3T3 fusion protein with Hsp90α and Cdc37 proteins. These interactions were further validated by reciprocal IPs followed by Western blotting. Activation of many kinases, such as FGFRs, depends on their interaction with the Hsp90 molecular chaperone system. Hsp90 recruitment is mediated by the co-chaperone adaptor protein Cdc37, which simultaneously binds to both the kinase and Hsp90. To test inhibition of F3T3 association with the Hsp90-Cdc37 complex, we treated U251 cells constitutively expressing F3T3 with the Hsp90 inhibitor Onalespib. We show that Onalespib treatment at 26 nM significantly suppresses the proliferation of F3T3 expressing U251 cells in comparison to micromolar levels of the pan-FGFR inhibitor BGJ398 and PD173074. Our results provide evidence that the F3T3 fusion gene contributes to drug resistance via multiple chemo-resistance pathways. Importantly, our findings establish that F3T3 is an Hsp90 client that shows strong addiction to the Hsp90-Cdc37 complex for cell growth and suggests a novel strategy for targeting F3T3 fusion gene in glioma. Citation Format: Tao Li, Farideh Mehraein-Ghomi, Sanjeev V. Namjoshi, Lynette M. Phillips, Elizabeth A. Ballard, Mary E. Forbes, ping-Chieh Chou, Xuejun Yang, Wei Zhang. Targeting Hsp90-Cdc37 complex overcomes drug resistance in glioma cells harboring FGFR3-TACC3 fusion gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1736.

Abstract: Ovarian cancer is the leading cause of cancer-related death among women. Complete cytoreductive surgery followed by platinum-taxene chemotherapy has been the gold standard for a long time. Various compounds have been assessed in an attempt to combine them with conventional chemotherapy to improve survival rates or even overcome chemoresistance. Many studies have shown that an antidiabetic drug, metformin, has cytotoxic activity in different cancer models. However, the synergism of metformin as a neoadjuvant formula plus chemotherapy in clinical trials and basic studies remains unclear for ovarian cancer. Methods We applied two clinical databases to survey metformin use and ovarian cancer survival rate. The Cancer Genome Atlas dataset, an L1000 microarray with Gene Set Enrichment Analysis (GSEA) analysis, Western blot analysis and an animal model were used to study the activity of the AKT/mTOR pathway in response to the synergistic effects of neoadjuvant metformin combined with chemotherapy. Results We found that ovarian cancer patients treated with metformin had significantly longer overall survival than patients treated without metformin. The protein profile induced by low- concentration metformin in ovarian cancer predominantly involved the AKT/mTOR pathway. In combination with chemotherapy, the neoadjuvant metformin protocol showed beneficial synergistic effects in vitro and in vivo. Conclusions This study shows that neoadjuvant metformin at clinically relevant dosages is efficacious in treating ovarian cancer, and the results can be used to guide clinical trials.