Zenggang Li

Down-regulation of 14-3-3ζ suppresses anchorage-independent growth of lung cancer cells through anoikis activation

The family of 14-3-3 proteins has emerged as critical regulators of diverse cellular responses under both physiological and pathological conditions. Here, we report an important role of 14-3-3ζ in tumorigenesis through a mechanism that involves anoikis resistance. 14-3-3ζ is up-regulated in a number of cancer types, including lung cancer. Through an RNAi approach using human lung adenocarcinoma-derived A549 cells as a model system, we have found that knockdown of a single ζ isoform of 14-3-3 is sufficient to restore the sensitivity of cancer cells to anoikis and impair their anchorage-independent growth. Enhanced anoikis appears to be mediated in part by up-regulated BH3-only proteins, Bad and Bim, coupled with decreased Mcl-1, resulting in the subsequent activation of Bax. This study suggests a model in which anchorage-independent growth of lung cancer cells requires the presence of 14-3-3ζ. This work not only reveals a critical role of 14-3-3ζ in anoikis suppression in lung cancer cells, but also identifies and validates 14-3-3ζ as a potential molecular target for anticancer therapeutic development.

The OncoPPi network of cancer-focused protein–protein interactions to inform biological insights and therapeutic strategies

As genomics advances reveal the cancer gene landscape, a daunting task is to understand how these genes contribute to dysregulated oncogenic pathways. Integration of cancer genes into networks offers opportunities to reveal protein–protein interactions (PPIs) with functional and therapeutic significance. Here, we report the generation of a cancer-focused PPI network, termed OncoPPi, and identification of >260 cancer-associated PPIs not in other large-scale interactomes. PPI hubs reveal new regulatory mechanisms for cancer genes like MYC, STK11, RASSF1 and CDK4. As example, the NSD3 (WHSC1L1)–MYC interaction suggests a new mechanism for NSD3/BRD4 chromatin complex regulation of MYC-driven tumours. Association of undruggable tumour suppressors with drug targets informs therapeutic options. Based on OncoPPi-derived STK11-CDK4 connectivity, we observe enhanced sensitivity of STK11-silenced lung cancer cells to the FDA-approved CDK4 inhibitor palbociclib. OncoPPi is a focused PPI resource that links cancer genes into a signalling network for discovery of PPI targets and network-implicated tumour vulnerabilities for therapeutic interrogation.

Molecular characterisation of formalin-fixed paraffin-embedded (FFPE) breast tumour specimens using a custom 512-gene breast cancer bead array-based platform

Background:Formalin-fixed, paraffin-embedded (FFPE) tumour tissue represents an immense but mainly untapped resource with respect to molecular profiling. The DASL (cDNA-mediated Annealing, Selection, extension, and Ligation) assay is a recently described, RT–PCR-based, highly multiplexed high-throughput gene expression platform developed by Illumina specifically for fragmented RNA typically obtained from FFPE specimens, which enables expression profiling. In order to extend the utility of the DASL assay for breast cancer, we have custom designed and validated a 512-gene human breast cancer panel.Methods:The RNA from FFPE breast tumour specimens were analysed using the DASL assay. Breast cancer subtype was defined from pathology immunohistochemical (IHC) staining. Differentially expressed genes between the IHC-defined subtypes were assessed by prediction analysis of microarrays (PAM) and then used in the analysis of two published data sets with clinical outcome data.Results:Gene expression signatures on our custom breast cancer panel were very reproducible between replicates (average Pearsons R2=0.962) and the 152 genes common to both the standard cancer DASL panel (Illumina) and our breast cancer DASL panel were similarly expressed for samples run on both panels (average R2=0.877). Moreover, expression of ESR1, PGR and ERBB2 corresponded well with their respective pathology-defined IHC status. A 30-gene set indicative of IHC-defined breast cancer subtypes was found to segregate samples based on their subtype in our data sets and published data sets. Furthermore, several of these genes were significantly associated with overall survival (OS) and relapse-free survival (RFS) in these previously published data sets, indicating that they are biomarkers of the different breast cancer subtypes and the prognostic outcomes associated with these subtypes.Conclusion:We have demonstrated the ability to expression profile degraded RNA transcripts derived from FFPE tissues on the DASL platform. Importantly, we have identified a 30-biomarker gene set that can classify breast cancer into subtypes and have shown that a subset of these markers is prognostic of OS and RFS.

Cables1 Complex Couples Survival Signaling to the Cell Death Machinery

Cables1 is a candidate tumor suppressor that negatively regulates cell growth by inhibiting cyclin-dependent kinases. Cables1 expression is lost frequently in human cancer but little is known about its regulation. Here, we report that Cables1 levels are controlled by a phosphorylation and 14-3-3-dependent mechanism. Mutagenic analyses identified two residues, T44 and T150, that are specifically critical for 14-3-3 binding and that serve as substrates for phosphorylation by the cell survival kinase Akt, which by binding directly to Cables1 recruits 14-3-3 to the complex. In cells, Cables1 overexpression induced apoptosis and inhibited cell growth in part by stabilizing p21 and decreasing Cdk2 kinase activity. Ectopic expression of activated Akt (AKT1) prevented Cables1-induced apoptosis. Clinically, levels of phosphorylated Cables1 and phosphorylated Akt correlated with each other in human lung cancer specimens, consistent with pathophysiologic significance. Together, our results illuminated a dynamic regulatory system through which activated Akt and 14-3-3 work directly together to neutralize a potent tumor suppressor function of Cables1.

Enabling systematic interrogation of protein–protein interactions in live cells with a versatile ultra-high-throughput biosensor platform

Large-scale genomics studies have generated vast resources for in-depth understanding of vital biological and pathological processes. A rising challenge is to leverage such enormous information to rapidly decipher the intricate protein-protein interactions (PPIs) for functional characterization and therapeutic interventions. While a number of powerful technologies have been employed to detect PPIs, a singular PPI biosensor platform with both high sensitivity and robustness in a mammalian cell environment remains to be established. Here we describe the development and integration of a highly sensitive NanoLuc luciferase-based bioluminescence resonance energy transfer technology, termed BRET(n), which enables ultra-high-throughput (uHTS) PPI detection in live cells with streamlined co-expression of biosensors in a miniaturized format. We further demonstrate the application of BRET(n) in uHTS format in chemical biology research, including the discovery of chemical probes that disrupt PRAS40 dimerization and pathway connectivity profiling among core members of the Hippo signaling pathway. Such hippo pathway profiling not only confirmed previously reported PPIs, but also revealed two novel interactions, suggesting new mechanisms for regulation of Hippo signaling. Our BRET(n) biosensor platform with uHTS capability is expected to accelerate systematic PPI network mapping and PPI modulator-based drug discovery.

Downregulation of urea transporter UT-A1 activity by 14-3-3 protein

Urea transporter (UT)-A1 in the kidney inner medulla plays a critical role in the urinary concentrating mechanism and thereby in the regulation of water balance. The 14-3-3 proteins are a family of seven isoforms. They are multifunctional regulatory proteins that mainly bind to phosphorylated serine/threonine residues in target proteins. In the present study, we found that all seven 14-3-3 isoforms were detected in the kidney inner medulla. However, only the 14-3-3 γ-isoform was specifically and highly associated with UT-A1, as demonstrated by a glutathione-S-transferase-14-3-3 pulldown assay. The cAMP/adenylyl cyclase stimulator forskolin significantly enhanced their binding. Coinjection of 14-3-3γ cRNA into oocytes resulted in a decrease of UT-A1 function. In addition, 14-3-3γ increased UT-A1 ubiquitination and protein degradation. 14-3-3γ can interact with both UT-A1 and mouse double minute 2, the E3 ubiquitin ligase for UT-A1. Thus, activation of cAMP/PKA increases 14-3-3γ interactions with UT-A1 and stimulates mouse double minute 2-mediated UT-A1 ubiquitination and degradation, thereby forming a novel regulatory mechanism of urea transport activity.

Abstract 1892: Investigating the role of Aurora Kinase A as a positive regulator of MAPK signaling

Aurora Kinase A (Aurora A), a protein canonically known to facilitate mitosis, has emerged as a promising drug target for cancer therapy. Aurora A is amplified in several cancer types, including glioblastoma, breast, and ovarian cancer, and increased expression is correlated to a worse prognosis for patients. In spite of these observations, the impact of increased Aurora A expression on cell signaling and cancer development remains unclear. Aurora A is also mis-localized to the cytoplasm in cancer and engages in oncogenic functions mediated through protein-protein interactions (PPIs). Thus, we hypothesized that through novel PPIs, Aurora A may be promoting oncogenic signaling and cancer cell growth. The acquired ability of cancer cells to sustain proliferative signaling is a cancer hallmark. One critical pathway through which mutations drive the development of several cancer types is the Ras-Raf-MEK-ERK signaling cascade, which is also known as the mitogen-associated protein kinase (Ras-MAPK) pathway. When we co-expressed Aurora A and H-Ras in both HEK-293T cells and the glioblastoma cancer cell line, 8-MG-BA, ERK phosphorylation increased compared to expression of Aurora A or H-Ras alone. However, pharmacological inhibition of Raf-1 and MEK was able to block the increase in ERK phosphorylation by Aurora A and H-Ras co-expression. In addition, Aurora A was not able to promote ERK phosphorylation when co-expressed with dominant negative H-Ras (S17N mutant). Together, these findings demonstrate that Aurora A requires the MAPK signaling cascade to potentiate ERK activation. Interestingly, we discovered that this effect correlates to a novel interaction between Aurora A and H-Ras. The Aurora A/H-Ras interaction was confirmed using the lysate-based assay, Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET), the affinity-based assay, GST-pull down, and in live cells using the Venus Protein-fragment Complementation Assay (PCA). Through deletion analysis, the binding domains critical to mediate the interaction of Aurora A and H-Ras were characterized. We also determined that H-Ras is not an enzymatic substrate for Aurora A using an in vitro kinase assay and found that cells treated with an Aurora A kinase inhibitor are able to maintain the Aurora A/H-Ras the interaction, suggesting that the Aurora A/H-Ras interaction is kinase-independent. In conclusion, our studies reveal a role for Aurora A as a positive regulator of Ras-MAPK proliferative signaling. As Aurora A gene expression is downstream of the Ras-MAPK signaling pathway, our data also provides a mechanism by which Aurora A forms a positive feedback loop, linking Aurora A to sustained proliferative signaling in cancer cells. Finally, the kinase-independence of the Aurora A/H-Ras interaction underscores the consideration that Aurora A kinase inhibitors currently in clinical development may not be sufficient to block all oncogenic functions of Aurora A. Citation Format: MaKendra Umstead, Jinglin Xiong, Zenggang Li, Andrei Ivanov, Yuhong Du, Haian Fu. Investigating the role of Aurora Kinase A as a positive regulator of MAPK signaling. [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 1892.

Abstract PR15: Predicting the essentialities of protein-protein interactions in cancer

Many protein-protein interactions (PPIs) are essential to cellular proliferation and survival, and thus represent potential targets for novel compounds with high specificity and therapeutic potential in the treatment of cancer. Despite their therapeutic significance, there are currently no high-throughput experimental platforms to interrogate the essentiality of individual protein-protein interactions. The ability to computationally predict or infer PPI essentiality would help prioritize the development of therapeutic targets and advance understanding of cancer biology, and remains a significant barrier. We have developed a computational method to predict PPI essentiality by combining shRNA studies with network models of protein interaction pathways in an analytic framework. High-throughput single-gene shRNA silencing is a well-established experimental approach to study protein essentiality in genome-wide screens. The silencing of a single gene in an shRNA screen effectively disrupts multiple PPIs, masking their individual contributions to the observed protein essentiality. Our method uses a network model to infer cliques of PPIs that are disrupted as each gene is silenced, then uses these sets to formulate an additive model of how the unknown PPI essentialities combine to form the observed protein essentialities. This process is then deconvoluted to recover the unknown essentialities of each PPI using a regularized solver. We performed this analysis in 108 cell lines characterized for protein essentiality in Project Achilles [1, 2]. A superpathway of 2186 proteins and 11488 protein interactions was constructed from prior knowledge databases [3] and protein interaction screenings. We demonstrate the validity of our approach via prediction of essential PPIs such as TP53-MDM4 in cell lines sensitive to nutlin and CDK4-RB1 interactions in cell lines sensitive to CDK4 inhibitors. [1] Cowley, Weir & Vazquez, et al. Nature Scientific Data 1, Article number: 140035. September 30, 2014 [2] Cheung HW, Cowley GS, Weir BA, et al. Proc Natl Acad Sci. 2011 Jul 26; 108(30):12372-7. [3] Schaefer CF, Anthony K, Krupa S, et al. Nucleic Acids Research. Jan 2009. 37:674-9 Citation Format: Lee AD Cooper, Josue D. Moran, Zenggang Li, Yuhong Du, Sahar Harati, Andrey A. Ivanov, Phillip Webber, Jonathan J. Havel, Margaret A. Johns, Haian Fu, Carlos S. Moreno. Predicting the essentialities of protein-protein interactions in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr PR15.

Abstract 112: An endogenous mTOR inhibitor, PRAS40, is upregulated in lung cancer and contributes to tumor cell invasion and chemoresistance

The proline-rich Akt substrate of 40-kDa (PRAS40) has recently been identified as a regulatory component of, TORC1 (a complex composed of mTOR, GβL, DEPTOR, RAPTOR and PRAS40)Phosphorylation of Thr246 on PRAS40 by PKB/Akt, and Ser183 and Ser221 by mTOR results in the dissociation of PRAS40 from TORC1, and binding of PRAS40 to 14-3-3 proteins. Negative mTOR regulators usually play anti-tumorigenesis roles (like TSC1-TSC2, LKB1 and PTEN). Unexpectedly, PRAS40 has been shown to acted as a pro-tumorigenic factor in certain types of cancer cells. To better understand the role of PRAS40 in cancer, we have carried out a series of cell culture and patient tumor tissue based studies. Here, we report that PRAS40 was upregulated in most of the non-small cell lung cancer (NSCLC) cell lines examined when compared to normal lung epithelial cell lines. Further immonuhistochemistry (IHC) studies confirmed the overexpression of PRAS40 in tumor tissues from NSCLC patients. Significantly, nearly 60% of cancer tissues from NSCLC patients exhibited considerably higher levels of PRAS40 protein than those in paired normal lung tissue. Such high levels of PRAS40 expression appear to be required for tumor progression as silencing of PRAS40 by shRNA decreased the cell invasion ability of NSCLC cells. Subsequently, we investigated whether PRAS40 can influence the sensitivity of NSCLC cells to chemotharepitic drugs. Our data demonstrated that downregulation of PRAS40 protein increased the sensitivity of NSCLC cells to chemotherapeutic agents, such as etopside and perifosine. In summary, our results strongly support a pro-tumorigenic role for PRAS40 and suggest PRAS40 could be a potential NSCLC therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 112. doi:1538-7445.AM2012-112

Abstract 4986: Nuclear PRAS40 regulates the cellular response to nucleolar stress

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The Proline-rich Akt Substrate of 40kDA (PRAS40) has recently been identified as a binding partner and inhibitor of the mammalian Target of Rapamycin Complex 1 (mTORC1), a growth factor- and nutrient-sensitive kinase whose activity promotes protein synthesis and cell growth. Despite its inhibitory effect on mTORC1, PRAS40 has been shown to promote cell survival in rodent models of spinal cord injury and tumorigenesis. Importantly, PRAS40 levels have also been found to correlate with poor prognosis in lung cancer patients, an effect not readily explained by mTORC1 inhibition. Here we demonstrate that in addition to its known cytoplasmic role in inhibiting mTORC1, PRAS40 also actively shuttles to the nucleus, where it congregates at the periphery of nucleoli and exists in a high-molecular weight complex void of mTORC1 components. Through immunoprecipitation and mass spectrometry analyses we have begun to identify members of this nuclear PRAS40 complex. Furthermore, we demonstrate that PRAS40 overexpression attenuates Actinomycin D-mediated induction of nucleolar stress markers. These findings may help to explain the apparent pro-tumorigenic effects of PRAS40 and identify the PRAS40 nuclear complex as a potential target for anti-cancer drug discovery. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4986. doi:1538-7445.AM2012-4986