Douglas Cress

Impact of upfront cellular enrichment by laser capture microdissection on protein and phosphoprotein drug target signaling activation measurements in human lung cancer: Implications for personalized medicine.

The aim of this study was to evaluate whether upfront cellular enrichment via laser capture microdissection (LCM) is necessary for accurately quantifying predictive biomarkers in nonsmall cell lung cancer tumors.

IKBKE Is a Substrate of EGFR and a Therapeutic Target in Non-Small Cell Lung Cancer with Activating Mutations of EGFR.

Non-small cell lung cancers (NSCLC) marked by EGFR mutations tend to develop resistance to therapeutic EGFR inhibitors, often due to secondary mutation EGFR(T790M) but also other mechanisms. Here we report support for a rationale to target IKBKE, an IκB kinase family member that activates the AKT and NF-κB pathways, as one strategy to address NSCLC resistant to EGFR inhibitors. While wild-type and mutant EGFR directly interacted with IKBKE, only mutant EGFR phosphorylated IKBKE on residues Y153 and Y179. The unphosphorylatable mutant IKBKE-Y153F/Y179-F that lost kinase activity failed to activate AKT and inhibited EGFR signaling. In clinical specimens of NSCLC with activating mutations of EGFR, we observed elevated levels of phospho-Y153 IKBKE. IKBKE ablation with shRNA or small-molecule inhibitor amlexanox selectively inhibited the viability of NSCLC cells with EGFR mutations in vitro In parallel, we found that these treatments activated the MAPK pathway due to attenuation of an IKBKE feedback mechanism. In vivo studies revealed that combining amlexanox with MEK inhibitor AZD6244 significantly inhibited the xenograft tumor growth of NSCLC cells harboring activating EGFR mutations, including EGFR(T790M) Overall, our findings define IKBKE as a direct effector target of EGFR and provide a therapeutic rationale to target IKBKE as a strategy to eradicate EGFR-TKI-resistant NSCLC cells. Cancer Res; 76(15); 4418-29. ©2016 AACR.

The retinoblastoma protein: a master tumor suppressor acts as a link between cell cycle and cell adhesion

RB1 was the first tumor suppressor gene discovered. Over four decades of work have revealed that the Rb protein (pRb) is a master regulator of biological pathways influencing virtually every aspect of intrinsic cell fate including cell growth, cell-cycle checkpoints, differentiation, senescence, self-renewal, replication, genomic stability and apoptosis. While these many processes may account for a significant portion of RB1s potency as a tumor suppressor, a small, but growing stream of evidence suggests that RB1 also significantly influences how a cell interacts with its environment, including cell-to-cell and cell-to-extracellular matrix interactions. This review will highlight pRbs role in the control of cell adhesion and how alterations in the adhesive properties of tumor cells may drive the deadly process of metastasis.

Abstract 1890: IKBKE is a substrate of EGFR and a therapeutic target in NSCLCs with activating mutations of EGFR

EGFR is one of key driver pathways of non-small cell lung cancer (NSCLC). EGFR inhibitors have shown significant response in patients with EGFR mutation. However, patients develop resistance in short period of time, which results from secondary EGFRT790M mutation and other mechanisms. Here, we demonstrated that activating mutations of EGFR significantly activated IKBKE, an IκB kinase family member previously shown to activate Akt and NF-κB pathways. Furthermore, we showed that EGFR directly interacts with and phosphorylates IKBKE on tyrosine 153 and tyrosine 179 residues. IKBKE-Y153F/179-F, a mutant that could not be phosphorylated by EGFR, largely reduced its kinase activity and failed to activate Akt and NF-κB as well as inhibited EGFR signaling. Furthermore, phospho-IKBKE-Y153 correlated with EGFR activation in NSCLC patients. Notably, depletion of IKBKE by either small molecule inhibitor amlexanox or shRNA selectively inhibited cell viability in NSCLC cells with EGFR mutations. Moreover, we observed that inhibition of IKBKE led to feedback activation of the MAPK pathway. Combination of amlexanox with MEK inhibitor AZD6244 significantly inhibits cell survival and tumor growth in NSCLC cells driven by activating EGFR mutations including EGFRT790M. Thus, our findings not only identify IKBKE as a direct downstream target of EGFR but also provide a potential therapeutic strategy for EGFR-TKI resistant NSCLC driven by secondary EGFR mutation. Citation Format: Sridevi Challa, Jian-Ping Guo, Cheng-xiong Xu, Yajuan Li, Donghwa Kim, Douglas Cress, Eric Haura, Domenico Coppola, Jin Cheng. IKBKE is a substrate of EGFR and a therapeutic target in NSCLCs with activating mutations of EGFR. [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 1890.

Abstract 2414: Overexpression of PAXIP1 potentiates WEE1 inhibitor action in lung cancer cells

Cells have developed extensive networks and pathways to detect and repair DNA at the sites of damage. The DNA damage response (DDR) involves a complex cascade of events and many proteins containing BRCA1 C-terminal (BRCT) modular domains are involved in this process. These domains are known to bind phosphopeptides and can occur as singleton or as tandem domains (tBRCT). Previously in our lab, 7 tBRCT containing proteins were used to perform a systematic analysis of protein-protein interactions (PPIN) in the DDR network. 18 kinases were found to be interactors in this network. We hypothesized that the kinases contained in this DDR network would be targets for sensitization to standard chemotherapy. We extracted the 18 kinases and conducted systematic pharmacological and siRNA based screens to evaluate the effect of inhibition of these kinases in combination with platinum-based agents. These screens revealed that inhibition of WEE1, a kinase known to be a negative regulator of mitosis has synergistic effects in combination with cisplatin in various lung cancer cell lines. In the tBRCT network, WEE1 interacted with PAXIP1, a tBRCT-containing protein that forms foci at sites of DNA damage and is required for cells to progress to mitosis. Therefore, the PAXIP1-WEE1 interaction in combination with results from our inhibitor screen prompted us to evaluate the role of PAXIP1 in WEE1 inhibitor treatment of lung cancer cell lines. AZD1775 (formerly, MK-1775) is a WEE1 inhibitor currently in clinical trials for various tumor types. We evaluated the levels of WEE1 expression in 15 lung cancer cell lines and observed that cell lines with high WEE1 levels have a better response to AZD1775 than cell lines that express low levels of WEE1. Furthermore, PAXIP1 levels are directly related to the cellular response to AZD1775 alone or in combination with cisplatin. Overexpressing PAXIP1 in lung cancer cell lines leads to an increased mitotic index upon WEE1 inhibition at the G2/M checkpoint. Overexpression of PAXIP1 also leads to increased levels of apoptosis when cells are treated with AZD1775 compared to cells with low levels of PAXIP1. We are currently evaluating the expression levels of PAXIP1 and WEE1 in lung tumors using tissue microarrays and exploring the mechanisms by which PAXIP1 regulates WEE1. Overall, our results indicate that PAXIP1 modulates the efficacy of WEE1 inhibition by AZD1775 and could be a potential biomarker of AZD1775 response. Citation Format: Ankita Jhuraney, Nicholas T. Woods, Fumi Kinose, Douglas W. Cress, Jhanelle E. Gray, Eric B. Haura, Uwe Rix, Alvaro N. Monteiro. Overexpression of PAXIP1 potentiates WEE1 inhibitor action in lung cancer cells. [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 2414. doi:10.1158/1538-7445.AM2014-2414

Abstract 206: Molecular mechanisms of aggressiveness of osteosarcomas.

Osteosarcomas (OS) are highly aggressive neoplasms of osteoblastic origin. Evidencing their early proclivity to metastasize, at the time of diagnosis most OS are already classified as high grade and poorly differentiated malignancies and about 20% have detectable metastases. Prognosis is very poor with only about 10% of patients achieving long-term disease-free intervals. In spite of this, OS aggressivity still remains to be mechanistically explained. Our work sheds light into this issue by uncovering a novel function for the retinoblastoma protein (Rb), a cell cycle repressor mutated in most OS, in promoting cell adhesion. Our microarrays comparing Rb-expressing with Rb-deficient osteoblasts showed that Rb regulates a wide variety of cell adhesion genes, specifically up-regulating the expression of osteoblast -specific cadherins and integrins, while down-regulating the expression of several metastasis-associated cell adhesion molecules. Cellular pathway analyses showed that 8 of the top 10 cellular processes affected by Rb are related to cell adhesion, with integrin- and adherens junctions-mediated adhesion having the first and fourth best p-values in the list, respectively. This regulation was confirmed when we re-introduced Rb into the Rb-null OS cell line Saos-2 and found an Rb-engendered change in the expression profile of several cadherins and integrins. Rb also strongly induced Integrin α10 (Itga10) in MC3T3 osteoblasts, and assays using Itga10 promoter/luciferase constructs showed that this induction is transcriptional and independent of Rb9s phosphorylation status. Point mutagenesis and progressive deletion of the Itga10 promoter allowed us to identify an Rb-responsive region from -108 to -55 bp relative to the transcriptional start site. We also characterized a second mechanism linking Rb with cell adhesion. This mechanism requires the Rb-mediated repression of Pak1, a Rac1-binding kinase that destabilizes cell adhesion when upregulated during carcinogenesis. Rb transcriptionally represses Pak1, as demonstrated by our nuclear run-on assays. Our Chip analyses showed an Rb-E2F complex binding to an Rb-responsive site in the Pak1 promoter that is rich in E2F binding sites, showing that Pak1 is an E2F target and suggesting that Rb9s repressive effect on Pak1 consists of blocking E2F9s transactivating activity on Pak1. Taken together, our results show that Rb promotes cell adhesion by inducing the expression of the cadherins and integrins necessary for cell adhesion to other cells and to a substrate, and by antagonizing the E2F-induced expression of cell adhesion destabilizers such as Pak1. Based on our data, we propose that Rb loss in OS exacerbates aggressiveness by debilitating cellular adhesion, which in turn facilitates tumor cell detachment and metastasis. Citation Format: Brienne Engel, Pedro G. Santiago-Cardona, Bernadete Sosa-Garcia, Douglas Cress. Molecular mechanisms of aggressiveness of osteosarcomas. [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 206. doi:10.1158/1538-7445.AM2013-206

Abstract C12: Molecular mechanisms of aggressiveness of Rb-deficient tumors

Osteosarcomas (OS) and small cell lung carcinomas (SCLC) are poorly differentiated, highly aggressive tumors, with an early proclivity to metastasize. Prognosis is poor for both, with only 10-20% of patients achieving long-term disease-free intervals. Still, OS and SCLC aggressivity remain to be molecularly explained. Interestingly, these cancer types show high rates of inactivation of the RB1 gene coding for the retinoblastoma protein (Rb), a known cell cycle repressor. This observation led us to hypothesize that in OS and SCLC, Rb loss during early tumorigenesis facilitates both increased proliferation and an early tendency for metastasis. Supporting our hypothesis, our previous studies in osteoblasts showed that Rb transcriptionally regulates a wide repertoire of cell adhesion genes, including those coding for adherens junction cadherins and integrins. We also found that adhrens junction assembly requires the Rb-dependent transcriptional repression of Pak1, a Rac1-binding kinase that destabilizes cell adhesion when up-regulated. Thus, the early proclivity for metastasis of OS and SCLC may be related to perturbations in cell adhesion due to Rb loss. Here we present new data using the SCLC line H187 and the non-small cell lung carcinoma (NSCLC) cell lines H1975 and H520. SCLC carcinomas have high rates (> 90%) of Rb loss while NSCLC are known to be Rb wild type. Therefore, comparing H1975 and H520 vs. H187 provided us with an Rb-proficient vs. Rb-deficient system similar to the one we previously used to study Rb-engendered effects in osteoblasts. Our immunoblots and qRT-PCR showed that the Rb-null H187 cells expressed dramatically reduced levels of E-cadherin mRNA and protein relative to H1975 and H520, confirming that Rb loss impairs cell adhesion due to adherens junction loss. Interestingly, N-cadherin is up-regulated in H187 cells, suggesting that Rb loss triggers a cadherin switch similar to the one associated with epithelial-to-mesenchymal transitions. Other cell adhesion-related genes we found down-regulated in Rb-null H187 cells relative to H1975 and H520, as determined by qRT-PCR, were α-catenin, β-catenin, OB-cadherin, and the cytoskeletal adapter proteins eplin, vinculin, formin-1, and α-actinin. Our immunoblots also showed decreased levels of merlin, an adherens junction-interacting protein, in H187 cells. We also studied the expression of Rac1-binding proteins, given their involvement in cell adhesion. Our immunoblots and qRT-PCR showed reduced levels of IQGAP1 in H187 cells relative to H1975 and H520 cells. Given that IQGAP1 is a GTPase activating protein (GAP) that represses Rac1 by promoting its GDP-bound inactive state, we postulate that Rb represses Rac1 function via IQGAP1. Beta-2-chimaerin, another Rac1 GAP was also down regulated at the protein level in H187 cells. Taken together, our data in lung cell lines recapitulate most of our findings in osteoblasts, both in regards to the effect of Rb on cell adhesion and to the need for the Rb-mediated repression of Rac1 for the establishment of cell adhesion. Based on our data, we propose that Rb loss in OS and SCLC, not only leads to an increased proliferative capacity during early tumorigenesis, but also exacerbates aggressiveness of tumors at early stages by perturbing cellular adhesion, which in turn facilitates tumor cell detachment and metastasis. Citation Format: Jonathan Gonzalez-Flores, Ruth Cruz-Cosme, Yariana Rodriguez-Ortiz, Sarah Brady, Ricardo Fraticelli-Rosado, Brienne Engel, Douglas Cress, Pedro G. Santiago-Cardona. Molecular mechanisms of aggressiveness of Rb-deficient tumors. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C12.

Abstract 3105: Orphan nuclear receptor NR4A2 exhibits oncogenic activity in lung cancer cells.

LKB1 (also known as STK11) is a frequent target for mutations in lung cancer, however, the role of LKB1 loss in tumorigenesis is undefined. NR4A2, together with NR4A1, and NR4A3, constitute the nuclear receptor (NR) subfamily 4A (NR4A), which are orphan NRs lacking identified ligands. NR4A family has been implicated in cell cycle regulation, apoptosis, inflammation, metabolism and more recently in carcinogenesis. However, there are conflicting data for tumorigenesis as NR4A1 and NR4A3 act as tumor suppressors in leukemia mouse models, while NR4A2 appears to stimulate tumor cell growth in vitro. Our laboratory discovered that the CRTC1 gene was aberrantly activated in LKB1-null cancer cells, stimulating the transcription of cAMP/CREB targets including NR4A gene members. The aim of this study is to examine NR4A2 as a potential oncogene and therapeutic target under the regulation of CRTC1 in LKB1 wildtype and null lung cancer cells. We demonstrated here that somatic loss of LKB1 in tumor cells was associated with underphosphorylation and nuclear localization of CRTC1, this resulted in upregulated expression and stronger transcriptional activity of NR4A2 in LKB1 mutant lung cancer cells and clinical primary lung cancer samples. We confirmed that forskolin, which can activate adenylate cyclase then catalyze the transformation of ATP to cAMP, could induce CRTC1 dephosphorylation resulting in enhanced NR4A2 expression. We identified that activation of the CRTC gene family induced NR4A2 expression, whereas knockdown of CRTCs decreased the expression of NR4A2. Furthermore, short hairpin RNA-mediated down-regulation of NR4A2, results in attenuated lung cancer cell proliferation and colony formation in vitro and decreased the tumor sizes in vivo in nude mice. In summary, our data suggested a CRTC-mediated transcriptional regulatory mechanism for NR4A2 activity, and proposed an oncogenic role of NR4A2 in LKB1 null lung cancer cell lines. Citation Format: Chunxia Cao, Min Zhang, Ruli Gao, Douglas Cress, Zirong Chen, Lizi Wu, Maria Zajac Kaye, Frederic Kaye. Orphan nuclear receptor NR4A2 exhibits oncogenic activity in lung cancer cells. [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 3105. doi:10.1158/1538-7445.AM2013-3105

Abstract 4025: The retinoblastoma protein regulates cell adhesion: Implications for lung cancer progression

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The retinoblastoma (Rb) protein is a tumor suppressor commonly inactivated in human cancers. Our work has uncovered a role for Rb as a regulator of adherens junction assembly and cell-to-cell adhesion. This is a novel function since Rb is predominantly known as a cell cycle repressor. Our objective is to characterize the molecular mechanism by which Rb performs this function. We hypothesized that Rb controls the activity of known regulators of adherens junction assembly. Using qRT-PCR, immunoblots, and transcriptional assays with promoter-luciferase constructs, we found that Rb represses the expression of Pak1, which is a known effector of the small Rho GTPase Rac1. Notably, Rac1 is a well known regulator of adherens junction assembly whose increased activity in cancer is linked to tumor metastasis. We found that Pak1 repression by Rb is transcriptional and is E2F-dependent. Our chromatin immunoprecipitation assays showed that an Rb-E2F complex binds to an Rb-responsive element in the Pak1 promoter that is rich in E2F binding sites. This suggests that Pak1 is an E2F target and that Rbs repressive effect on Pak1 consists in blocking E2F activity. Further supporting a role for Rb in cell adhesion, microarray analyses comparing Rb-expressing with Rb-deficient cells showed that Rb transcriptionally regulates a variety of cell adhesion genes, including those coding for adherens junction cadherins. Pak1 also appeared among the transcript repressed by Rb, validating Rac1 and Pak1 as links between Rb and adherens junctions. Importantly, we have evidence suggesting that the de-regulation in cell adhesion-related gene expression due to Rb loss may be related to lung cancer development. We examined the Directors Challenge lung cancer database and found that numerous Rb-regulated cell adhesion genes correlate with overall survival in lung adenocarcinoma patients. For this analysis we arbitrarily defined an RB1 signature as all RB1-regulated genes with >2 activation or repression with statistically significant p-values. We found that a total of 1,154 RB1-regulated genes correlated with overall survival. This analysis suggests that a subset of RB1-regulated cell adhesion genes may be critical in the development of lung cancer. Three examples of RB1-activated cell adhesion genes that correlate with overall survival are E cadherin (Cdh 1), Integrin alpha 1 (Itga1) and Integrin alpha 10 (ItgA10). In summary, three main conclusions emerge from our studies. First, Rb transcriptionally controls expression of cadherins and other adherens junction-related genes. Second, Rb also controls adherens junction assembly by repressing Rac1 and its effector Pak1. Third, global de-regulation of cell adhesion due to Rb loss could be part of the molecular events associated to lung cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4025. doi:10.1158/1538-7445.AM2011-4025

Abstract 5048: Targeting regulators of Chk1 to enhance cytotoxic efficacy of HDAC inhibitors

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Predicting and overcoming drug resistance is a major difficulty facing the efficacy of drugs in clinical trials. Histone deacetylase inhibitors (HDACis) are known to induce cell death and have been used in clinic trials as anticancer drugs, however, the cytotoxic effects of HDACis remains to be limited with no clear mechanism of resistance has been elucidated to explain this limited efficacy. In this study we showed that downregulation of the G2 checkpoint kinase 1 (Chk1) plays an essential role in inducing cell death by HDACis. We identified E2F as a main regulator of Chk1 expression in cancer cells, suggesting that inhibition of Chk1 through E2F may enhance the cytotoxic effects of HDACis in clinical studies. Methods: A549, H1299, PC9, HCT116, HT1080, MNNG, H2172, Saos-2, MG63, U2OS, p21-/- and p21+/+ MEF cell lines were grown in appropriate medium and treated with the HDAC inhibitors, SaHa, LBH589 or MS275. Western blot and qRT-PCR experiments were performed to assess the effect of HDACis on Chk1/G2-M checkpoint pathways and on E2F-mediated gene expression profiles. Additionally, knock out and gain of function experiments were done to better understand the interaction between Chk1, E2F and p21Waf1 in HDACi mediated cell death. The results of the in vitro cell lines studies were corroborated in the ex vivo experiments with patient tumor tissue. Results: We showed that HDACi treatment leads to inhibition of Chk1 expression and consequently to increased activity of CDC25 and CDC2 (CDK1) enzymes causing premature mitotic entry and cell death. In the knock-down experiments we found that inhibition of E2F1 or E2F3 leads to reduction of Chk1 expression and potentiation of cell death by HDACis. Similar results were observed using patient-derived tumor samples treated with SaHa and an E2F inhibitor (HLM006474). Additionally, over-expression of E2F1 or E2F3 dramatically decreased HDACi-mediated cell death. In time course experiments, Chk1 as well as other E2F-responsive genes were down regulated after HDACi treatment accompanied by an expected p21Waf1 induction. In p21Waf1 +/+ and -/- MEFs we showed that lack of p21Waf1 results in increased apoptosis, suggesting that early induction of p21Waf1 by HDACis negatively regulates HDACi-induced cell death, which likely contributes to HDACi resistance in tumor cells by preventing cells to undergo mitotic cell death. Conclusions: Our results demonstrate that E2F-mediated downregulation of Chk1 plays a key role in HDACi-induced cell. We provide evidence that lack of Chk1 downregulation and activation of CDC2 is associated with resistance to HDACis indicating that Chk1 may represent a clinically relevant biomarker to assess the efficacy of HDACis in patient tumor samples. Taken together our data lends insight into the molecular mechanism of sensitivity and resistance to HDACi-induced cell death and will hopefully provide a stronger foundation for future clinical translation with HDACis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5048. doi:10.1158/1538-7445.AM2011-5048