Dunrui Wang

The Expression of Glyceraldehyde-3-Phosphate Dehydrogenase Associated Cell Cycle (GACC) Genes Correlates with Cancer Stage and Poor Survival in Patients with Solid Tumors

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is often used as a stable housekeeping marker for constant gene expression. However, the transcriptional levels of GAPDH may be highly up-regulated in some cancers, including non-small cell lung cancers (NSCLC). Using a publically available microarray database, we identified a group of genes whose expression levels in some cancers are highly correlated with GAPDH up-regulation. The majority of the identified genes are cell cycle-dependent (GAPDH Associated Cell Cycle, or GACC). The up-regulation pattern of GAPDH positively associated genes in NSCLC is similar to that observed in cultured fibroblasts grown under conditions that induce anti-senescence. Data analysis demonstrated that up-regulated GAPDH levels are correlated with aberrant gene expression related to both glycolysis and gluconeogenesis pathways. Down-regulation of fructose-1,6-bisphosphatase (FBP1) in gluconeogenesis in conjunction with up-regulation of most glycolytic genes is closely related to high expression of GAPDH in the tumors. The data presented demonstrate that up-regulation of GAPDH positively associated genes is proportional to the malignant stage of various tumors and is associated with an unfavourable prognosis. Thus, this work suggests that GACC genes represent a potential new signature for cancer stage identification and disease prognosis.

CDK5 is a major regulator of the tumor suppressor DLC1

CDK5 activates the tumor suppressor DLC1 by phosphorylating and diminishing the binding of an autoinhibitory region of DLC1 to its Rho-GAP domain and allows it to localize to focal adhesions.

Tumor-infiltrating myeloid cells activate Dll4/Notch/TGF-β signaling to drive malignant progression.

Myeloid cells that orchestrate malignant progression in the tumor microenvironment offer targets for a generalized strategy to attack solid tumors. Through an analysis of tumor microenvironments, we explored an experimental model of lung cancer that uncovered a network of Dll4/Notch/TGF-β1 signals that links myeloid cells to cancer progression. Myeloid cells attracted to the tumor microenvironment by the tumor-derived cytokines CCL2 and M-CSF expressed increased levels of the Notch ligand Dll4, thereby activating Notch signaling in the tumor cells and amplifying tumor-intrinsic Notch activation. Heightened Dll4/Notch signaling in tumor cells magnified TGF-β-induced pSMAD2/3 signaling and was required to sustain TGF-β-induced tumor cell growth. Conversely, Notch blockade reduced TGF-β signaling and limited lung carcinoma tumor progression. Corroborating these findings, by interrogating RNAseq results from tumor and adjacent normal tissue in clinical specimens of human head and neck squamous carcinoma, we found evidence that TGF-β/Notch crosstalk contributed to progression. In summary, the myeloid cell-carcinoma signaling network we describe uncovers novel mechanistic links between the tumor microenvironment and tumor growth, highlighting new opportunities to target tumors where this network is active.

Inactivation of the Dlc1 Gene Cooperates with Downregulation of p15INK4b and p16Ink4a, Leading to Neoplastic Transformation and Poor Prognosis in Human Cancer

The tumor suppressor gene deleted in liver cancer-1 (DLC1), which encodes a protein with strong RhoGAP (GTPase activating protein) activity and weak Cdc42GAP activity, is inactivated in various human malignancies. Following Dlc1 inactivation, mouse embryo fibroblasts (MEF) with a conditional Dlc1 knockout allele reproducibly underwent neoplastic transformation. In addition to inactivation of Dlc1 and increased activity of Rho and Cdc42, transformation depended on the subsequent decreased expression of the Cdk4/6 inhibitors p15(Ink4b) and p16(Ink4a) together with increased expression and activation of Cdk4/6. The level of expression of these cell-cycle regulatory genes was relevant to human tumors with low DLC1 expression. Analysis of publicly available annotated datasets of lung and colon cancer with gene expression microarray profiles indicated that, in pairwise comparisons, low DLC1 expression occurred frequently together (P < 0.01) with downregulation of p15(Ink4b) or p16(Ink4a) or upregulation of CDK4 or CDK6. In addition, an unfavorable prognosis (P < 0.05) was associated with low DLC1 and low p15(Ink4b) in lung cancer and colon cancer, low DLC1 and low p16(Ink4a) in lung cancer, low DLC1 and high CDK4 in lung cancer, and low DLC1 and high CDK6 in colon cancer. Thus, several genes and biochemical activities collaborate with the inactivation of DLC1 to give rise to cell transformation in MEFs, and the identified genes are relevant to human tumors with low DLC1 expression.

DLC1 is the principal biologically-relevant down-regulated DLC family member in several cancers

The RHO family of RAS-related GTPases in tumors may be activated by reduced levels of RHO GTPase accelerating proteins (GAPs). One common mechanism is decreased expression of one or more members of the Deleted in Liver Cancer (DLC) family of Rho-GAPs, which comprises three closely related genes (DLC1, DLC2, and DLC3) that are down-regulated in a wide range of malignancies. Here we have studied their comparative biological activity in cultured cells and used publicly available datasets to examine their mRNA expression patterns in normal and cancer tissues, and to explore their relationship to cancer phenotypes and survival outcomes. In The Cancer Genome Atlas (TCGA) database, DLC1 expression predominated in normal lung, breast, and liver, but not in colorectum. Conversely, reduced DLC1 expression predominated in lung squamous cell carcinoma (LSC), lung adenocarcinoma (LAD), breast cancer, and hepatocellular carcinoma (HCC), but not in colorectal cancer. Reduced DLC1 expression was frequently associated with promoter methylation in LSC and LAD, while DLC1 copy number loss was frequent in HCC. DLC1 expression was higher in TCGA LAD patients who remained cancer-free, while low DLC1 had a poorer prognosis than low DLC2 or low DLC3 in a more completely annotated database. The poorest prognosis was associated with low expression of both DLC1 and DLC2 (P < 0.0001). In cultured cells, the three genes induced a similar reduction of Rho-GTP and cell migration. We conclude that DLC1 is the predominant family member expressed in several normal tissues, and its expression is preferentially reduced in common cancers at these sites.

Abstract 1574: CDK5 negatively regulates Rho by phosphorylating and activating the Rho-GAP and tumor suppressor functions of DLC1

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The tumor suppressor Deleted in Liver Cancer 1 (DLC1) is frequently down-regulated in various human cancers, including breast, prostate, liver, and non-small cell lung cancer (NSCLC). Although the tumor suppressor function of DLC1 depends on its Rho-GAP (GTPase activating protein) activity, which converts Rho from the active GTP-bound form to the inactive GDP-bound form, the mechanisms regulating this activity are poorly understood. Here we determined that CDK5, a predominantly cytoplasmic serine/threonine kinase, negatively regulates Rho-GTP in normal and transformed cells that express DLC1, and that this regulation is attributable to the ability of CDK5 to activate DLC1. We found that CDK5, phosphorylates four serine residues (S120, S205, S422, S509) in the N-terminal region of DLC1, which reduces the binding of the N-terminus to the Rho-GAP domain. The phosphorylation of these serine residues stimulate the tumor suppressor activity of DLC1 - as determined by anchorage-independent growth and tumor xenograft formation - by increasing its Rho-GAP activity and facilitating its binding of several ligands. Serine-to-alanine mutations of the 4 serines increased binding between the DLC1 N-terminus and its Rho-GAP domain, resulting in a mutant protein that lacked these activities. We infer that the N-terminus of DLC1 functions as an auto-inhibitory domain that binds the Rho-GAP domain, and that phosphorylation of the N-terminal 4 serines by CDK5 reduces this interaction, activateing the Rho-GAP and tumor suppressor activities of DLC1. Interestingly, in NSCLC, the expression of CDK5, which has numerous targets, can be associated with a poor prognosis, suggesting that its activity in NSCLC might select for down-regulation of DLC1. Consistent with this hypothesis, we found that siRNA knock-down of endogenous DLC1 in a NSCLC line greatly increased the ability of CDK5 to stimulate neoplastic growth of the line. We also found, in a publicly available tumor database, that poorly differentiated NSCLC is much more likely to express high levels of CDK5 mRNA together with low levels of DLC1 mRNA than is well differentiated NSCLC. Thus, we have identified negative Rho regulation as a new function for CDK5, found DLC1 is the key target for this regulation, elucidated a CDK5-dependent post-translational modification for activating DLC1, and demonstrated the relevance of these observations to NSCLC. Citation Format: Brajendra K. Tripathi, Xiaolan Qian, Philipp Mertins, Dunrui Wang, Alex Papageorge, Steven Carr, Douglas R. Lowy. CDK5 negatively regulates Rho by phosphorylating and activating the Rho-GAP and tumor suppressor functions of DLC1. [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 1574. doi:10.1158/1538-7445.AM2014-1574

Effects of DLC1 Deficiency on Endothelial Cell Contact Growth Inhibition and Angiosarcoma Progression

Background Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene frequently deleted in cancer. However, DLC1 is not known to be deleted in angiosarcoma, an aggressive malignancy of endothelial cell derivation. Additionally, the physiologic functions of DLC1 protein in endothelial cells are poorly defined. Methods We investigated the effects of shRNA-induced DLC1 depletion in endothelial cells. Cell growth was measured by 3H thymidine incorporation, IncuCyte imaging, and population doublings; cell death by cell cycle analysis; gene expression by Affimetrix arrays and quantitative polymerase chain reaction; NF-κB activity by reporter assays; and protein levels by immunoblotting and immunofluorescence staining. We tested Tanespimycin/17-AAG and Fasudil treatment in groups of nine to 10 mice bearing ISOS-1 angiosarcoma. All statistical tests were two-sided. Results We discovered that DLC1 is a critical regulator of cell contact inhibition of proliferation in endothelial cells, promoting statistically significant (P < .001) cell death when cells are confluent (mean [SD] % viability: control DLC1 = 15.6 [19.3]; shDLC1 = 73.4 [13.1]). This prosurvival phenotype of DLC1-depleted confluent endothelial cells is attributable to a statistically significant and sustained increase of NF-κB activity (day 5, P = .001; day 8, P = .03) associated with increased tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) signaling. Consistently, we found that DLC1 is statistically significantly reduced (P < .001 in 5 of 6) and TNFAIP3/A20 is statistically significantly increased (P < .001 in 2 of 3 and P = 0.02 in 1 of 3) in human angiosarcoma compared with normal adjacent endothelium. Treatment with the NF-κB inhibitor Tanespimycin/17-AAG statistically significantly reduced angiosarcoma tumor growth in mice (treatment tumor weight vs control, 0.50 [0.19] g vs 0.91 [0.21] g, P = .001 experiment 1; 0.66 [0.26] g vs 1.10 [0.31] g, P = .01 experiment 2). Conclusions These results identify DLC1 as a previously unrecognized regulator of endothelial cell contact inhibition of proliferation that is depleted in angiosarcoma and support NF-κB targeting for the treatment of angiosarcoma where DLC1 is lost.

Receptor tyrosine kinase activation of RhoA is mediated by AKT phosphorylation of DLC1

We report several receptor tyrosine kinase (RTK) ligands increase RhoA–guanosine triphosphate (GTP) in untransformed and transformed cell lines and determine this phenomenon depends on the RTKs activating the AKT serine/threonine kinase. The increased RhoA-GTP results from AKT phosphorylating three serines (S298, S329, and S567) in the DLC1 tumor suppressor, a Rho GTPase-activating protein (RhoGAP) associated with focal adhesions. Phosphorylation of the serines, located N-terminal to the DLC1 RhoGAP domain, induces strong binding of that N-terminal region to the RhoGAP domain, converting DLC1 from an open, active dimer to a closed, inactive monomer. That binding, which interferes with the interaction of RhoA-GTP with the RhoGAP domain, reduces the hydrolysis of RhoA-GTP, the binding of other DLC1 ligands, and the colocalization of DLC1 with focal adhesions and attenuates tumor suppressor activity. DLC1 is a critical AKT target in DLC1-positive cancer because AKT inhibition has potent antitumor activity in the DLC1-positive transgenic cancer model and in a DLC1-positive cancer cell line but not in an isogenic DLC1-negative cell line.

Abstract 888: Inactivation of the DLC1 RhoGAP tumor suppressor by point mutation occurs commonly in human cancer and can result from Rho-dependent or Rho-independent mechanisms

The Rho GTPases, which are frequently activated in advanced cancer, stimulate their downstream effectors to regulate a variety of fundamental cellular process, e.g., cell cycle, cell migration, cell-cell adhesion, and cytoskeleton organization. The down-regulation of RhoGAPs is one mechanism of increased Rho activity, and members of the tumor suppressor DLC Rho-GAP family (DLC1-3) are frequently down-regulated by gene deletion or DNA methylation in human cancers. Here we report that, in addition, point mutations in the DLC1-3 coding regions occur frequently in certain cancers, most of the mutants we have analyzed have reduced tumor suppressor activity, at least two distinct mechanisms can account for this reduction, and one mechanism appears to be Rho-independent. In the TCGA database, DLC1 was mutated in 11% of gastric cancer (GAC), 10% colorectal cancer (CRC), 7% of melanomas (MEL), and 6% of lung adenocarcinoma (LAD). Most alterations were point mutations encoding a single amino acid change. Mutation of DLC2 and DLC3 also occurred in these tumor types, but were less frequent. The mutation frequency of p190-A, which is a Rho-GAP from a different gene family, was even less frequent, ranging from 4% in LAD to zero in GAC. Taken together, mutation of at least one DLC gene or p190-A was found in about 20% of LAD, CRC, and MEL, and 15% of GAC. In LAD, the DLC1 expression levels from the patients with DLC1 mutations were lower than the patients with DLC1 wild type, suggesting selection for decreased DLC1 expression in tumors harboring mutant DLC1. We constructed expression vectors for many of the DLC1 mutants and analyzed their phenotypes in human tumor cell lines. Most were found to be loss of tumor suppressor function mutants, as they were deficient for reducing growth in soft agar, cell migration, and focal adhesion turnover. One class of deficient mutants had lesions in the RhoGAP domain, which directly inactivated its RhoGAP activity and increased the activity of Rho downstream effectors. A second class had lesions in the DLC1 START (StAR-related lipid-transfer) domain, which is known to bind caveolin. although the RhoGAP activity was normal, complex formation with caveolin was much lower than with wild type DLC1. We conclude that, in addition to the frequent down-regulation of DLC1 expression via gene deletion or DNA methylation in tumors, point mutation of its coding sequence commonly inactivates the tumor suppressor activity of DLC1 at the protein level by at least two alternate mechanisms: attenuation of it binding to caveolin or of its RhoGAP activity, which up-regulates RhoA and its downstream effectors. (# Contributed equally) Citation Format: Xiaolan Qian, Dunrui Wang, Beatriz Sanchez-Solana, Brajendra K. Tripathi, Marian E. Durkin, Alex Papageorge, Douglas R. Lowy. Inactivation of the DLC1 RhoGAP tumor suppressor by point mutation occurs commonly in human cancer and can result from Rho-dependent or Rho-independent mechanisms. [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 888.

Abstract 4377: AKT positively regulates Rho-GTP by attenuating the GAP activity of the DLC1 tumor suppressor: a mechanistic study with translational implications

Many human cancers have high AKT activity and constitutive up-regulation of Rho-GTP, which is a major factor in the neoplastic process, but AKT is not known to regulate Rho-GTP. In this study, we show that AKT positively regulates Rho-GTP by directly phosphorylating tumor suppressor DLC1 and attenuating the Rho-GAP activity of DLC1, which catalyzes the conversion of active Rho-GTP to inactive Rho-GDP. Interestingly, AKT was found to increase Rho-GTP and its downstream activities in DLC1-positive cancer lines whether the DLC1 was endogenous or transfected, but not in DLC1-negative lines. Similarly, when epithelial cell lines were stimulated with EGF, they activated AKT, which increased Rho-GTP in a DLC1-dependent manner. Three Serines (S298, S329, S567) in DLC1 have AKT consensus motifs and were phosphorylated by AKT in vitro and in vivo. Their phosphorylation attenuated the Rho-GAP and tumor suppressor activities of DLC1 - including decreased cell migration, focal adhesion turnover, anchorage-independent growth, and tumor growth in mice - as did the combined phosphomimetic mutant DLC1-3D. By contrast, the combined Serine to Alanine mutant DLC1-3A was even more active than wild type DLC1 (DLC1-WT). Remarkably, an AKT inhibitor stimulated the tumor suppressor and Rho-GAP activities of DLC1-WT, but did not influence DLC1-3A or DLC1-3D. The N-terminal half of DLC1 (amino acids 1-600) bound the DLC1 Rho-GAP domain (amino acids 609-878), as determined by in vivo complex formation and by microscale thermophoresis, which measures protein interactions in close-to-native conditions. There was increased binding and decreased Rho-GAP activity when the three Serines in the N-terminus were phosphorylated or carried the 3D mutations. Conversely, there was decreased binding and increased Rho-GAP activity when the Serines were not phosphorylated or carried the 3A mutations. In a xenograft mouse model, AKT inhibitor treatment of DLC1-positive palpable tumors reduced their size, their Rho-GTP level, and their Rho-dependent signaling, but did not affect these three parameters in isogenic DLC1-negative tumors. Similarly, in the MMTV-PyMT breast cancer model, which had high AKT activity, high Rho-GTP, and expressed DLC1, an AKT inhibitor reduced the size of palpable tumors, and reduced Rho-GTP and its downstream signaling. AKT inhibition reduced DLC1 phosphorylation in both tumor models. We conclude: AKT can increase Rho-GTP by phosphorylating three N-terminal Serines in DLC1, which attenuates its Rho-GAP and tumor suppressor functions; the N-terminus of DLC1 is an auto-inhibitory domain that reversibly binds the Rho-GAP domain; AKT attenuates DLC1 functions by phosphorylating the Serines in the N-terminus, which increases its binding the Rho-GAP domain; and AKT inhibition has greater anti-tumor activity in DLC1-positive tumors than in DLC1-negative tumors. Citation Format: Brajendra K. Tripathi, Tiera Grant, Philipp Mertins, Xiaolan Qian, Dunrui Wang, Alex G. Papageorge, Steven A. Carr, Douglas R. Lowy. AKT positively regulates Rho-GTP by attenuating the GAP activity of the DLC1 tumor suppressor: a mechanistic study with translational implications. [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 4377.