Brienne E. Engel

Differential association of STK11 and TP53 with KRAS mutation-associated gene expression, proliferation and immune surveillance in lung adenocarcinoma.

While mutations in the KRAS oncogene are among the most prevalent in human cancer, there are few successful treatments to target these tumors. It is also likely that heterogeneity in KRAS-mutant tumor biology significantly contributes to the response to therapy. We hypothesized that the presence of commonly co-occurring mutations in STK11 and TP53 tumor suppressors may represent a significant source of heterogeneity in KRAS-mutant tumors. To address this, we utilized a large cohort of resected tumors from 442 lung adenocarcinoma patients with data including annotation of prevalent driver mutations (KRAS and EGFR) and tumor suppressor mutations (STK11 and TP53), microarray-based gene expression and clinical covariates, including overall survival (OS). Specifically, we determined impact of STK11 and TP53 mutations on a new KRAS mutation-associated gene expression signature as well as previously defined signatures of tumor cell proliferation and immune surveillance responses. Interestingly, STK11, but not TP53 mutations, were associated with highly elevated expression of KRAS mutation-associated genes. Mutations in TP53 and STK11 also impacted tumor biology regardless of KRAS status, with TP53 strongly associated with enhanced proliferation and STK11 with suppression of immune surveillance. These findings illustrate the remarkably distinct ways through which tumor suppressor mutations may contribute to heterogeneity in KRAS-mutant tumor biology. In addition, these studies point to novel associations between gene mutations and immune surveillance that could impact the response to immunotherapy.

Role of LKB1-CRTC1 on Glycosylated COX-2 and Response to COX-2 Inhibition in Lung Cancer

BACKGROUND Cyclooxygenase-2 (COX-2) directs the synthesis of prostaglandins including PGE-2 linking inflammation with mitogenic signaling. COX-2 is also an anticancer target, however, treatment strategies have been limited by unreliable expression assays and by inconsistent tumor responses to COX-2 inhibition. METHODS We analyzed the TCGA and Directors Challenge lung cancer datasets (n = 188) and also generated an LKB1-null lung cancer gene signature (n = 53) to search the Broad Institute/Connectivity-MAP (C-MAP) dataset. We performed ChIP analyses, real-time polymerase chain reaction, immunoblotting, and drug testing of tumor cell lines (n = 8) and primary lung adenocarcinoma surgical resections (n = 13). RESULTS We show that COX-2 is a target of the cAMP/CREB coactivator CRTC1 signaling pathway. In addition, we detected a correlation between LKB1 status, CRTC1 activation, and presence of glycosylated, but not inactive hypoglycosylated COX-2 in primary lung adenocarcinoma. A search of the C-MAP drug database discovered that all high-ranking drugs positively associated with the LKB1-null signature are known CRTC1 activators, including forskolin and six different PGE-2 analogues. Somatic LKB1 mutations are present in 20.0% of lung adenocarcinomas, and we observed growth inhibition with COX-2 inhibitors in LKB1-null lung cancer cells with activated CRTC1 as compared with LKB1-wildtype cells (NS-398, P = .002 and Niflumic acid, P = .006; two-tailed t test). CONCLUSION CRTC1 activation is a key event that drives the LKB1-null mRNA signature in lung cancer. We also identified a positive feedback LKB1/CRTC1 signaling loop for COX-2/PGE2 regulation. These data suggest a role for LKB1 status and glycosylated COX-2 as specific biomarkers that provide a framework for selecting patients for COX-2 inhibition studies.

Expression of integrin alpha 10 is transcriptionally activated by pRb in mouse osteoblasts and is downregulated in multiple solid tumors

pRb is known as a classic cell cycle regulator whose inactivation is an important initiator of tumorigenesis. However, more recently, it has also been linked to tumor progression. This study defines a role for pRb as a suppressor of the progression to metastasis by upregulating integrin α10. Transcription of this integrin subunit is herein found to be pRb dependent in mouse osteoblasts. Classic pRb partners in cell cycle control, E2F1 and E2F3, do not repress transcription of integrin α10 and phosphorylation of pRb is not necessary for activation of the integrin α10 promoter. Promoter deletion revealed a pRb-responsive region between −108 bp to −55 bp upstream of the start of the site of transcription. pRb activation of transcription also leads to increased levels of integrin α10 protein and a greater concentration of the integrin α10 protein at the cell membrane of mouse osteoblasts. These higher levels of integrin α10 correspond to increased binding to collagen substrate. Consistent with our findings in mouse osteoblasts, we found that integrin α10 is significantly underexpressed in multiple solid tumors that have frequent inactivation of the pRb pathway. Bioinformatically, we identified data consistent with an ‘integrin switch’ that occurs in multiple solid tumors consisting of underexpression of integrins α7, α8, and α10 with concurrent overexpression of integrin β4. pRb promotes cell adhesion by inducing expression of integrins necessary for cell adhesion to a substrate. We propose that pRb loss in solid tumors exacerbates aggressiveness by debilitating cellular adhesion, which in turn facilitates tumor cell detachment and metastasis.

A Sensitive NanoString-Based Assay to Score STK11 (LKB1) Pathway Disruption in Lung Adenocarcinoma

Introduction: Serine/threonine kinase 11 gene (STK11), better known as liver kinase &bgr;1, is a tumor suppressor that is commonly mutated in lung adenocarcinoma (LUAD). Previous work has shown that mutational inactivation of the STK11 pathway may serve as a predictive biomarker for cancer treatments, including phenformin and cyclooxygenase‐2 inhibition. Although immunohistochemical (IHC) staining and diagnostic sequencing are used to measure STK11 pathway disruption, there are serious limitations to these methods, thus emphasizing the importance of validating a clinically useful assay. Methods: An initial STK11 mutation mRNA signature was generated using cell line data and refined using three large, independent patient databases. The signature was validated as a classifier using The Cancer Genome Atlas (TCGA) LUAD cohort as well as a 442‐patient LUAD cohort developed at Moffitt. Finally, the signature was adapted to a NanoString‐based format and validated using RNA samples isolated from formalin‐fixed, paraffin‐embedded tissue blocks corresponding to a cohort of 150 patients with LUAD. For comparison, STK11 IHC staining was also performed. Results: The STK11 signature was found to correlate with null mutations identified by exon sequencing in multiple cohorts using both microarray and NanoString formats. Although there was a statistically significant correlation between reduced STK11 protein expression by IHC staining and mutation status, the NanoString‐based assay showed superior overall performance, with a –0.1588 improvement in area under the curve in receiver‐operator characteristic curve analysis (p < 0.012). Conclusion: The described NanoString‐based STK11 assay is a sensitive biomarker to study emerging therapeutic modalities in clinical trials.

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.

Early2 factor (E2F) deregulation is a prognostic and predictive biomarker in lung adenocarcinoma

Clinicians routinely prescribe adjuvant chemotherapy (ACT) for resected non-small cell lung cancer patients. However, ACT only improves five-year disease-free survival in stage I-III non-small cell lung cancer by 5-15%, with most patients deriving no benefit. Herein, deregulation of the E2F pathway was explored as a biomarker in lung adenocarcinoma patients. An E2F pathway scoring system, based on 74 E2F-regulated genes, was trained for RNA from two platforms: fresh-frozen (FF) or formalin-fixed paraffin-embedded (FFPE) tissues. The E2F score was tested as a prognostic biomarker in five FF-based cohorts and two FFPE-based cohorts. The E2F score was tested as a predictive biomarker in two randomized clinical trials; JBR10 and the NATCH (Neo-Adjuvant Taxol-Carboplatin Hope) trial. The E2F score was prognostic in untreated patients in all seven datasets examined (p < 0.05). Stage-specific analysis of combined cohorts demonstrated that the E2F score was prognostic in stage I patients (p = 0.0495 to <0.001; hazard ratio, HR, =2.04- 2.22) with a similar trend in other stages. The E2F score was strongly predictive in stage II patients from the two combined randomized clinical trials with a significant differential treatment effect (p = 0.015). Specifically, ACT improved survival in stage II patients with high E2F (p = 0.01; HR= 0.21). The 5-year survival increased from 18% to 81%. In contrast, in patients with low E2F, 5-year survival was 57% in untreated patients and 41% in ACT-treated patients with a HR of 1.55 (p = 0.47). In summary, the E2F score provides valuable prognostic information for Stage I and predictive information for Stage II lung adenocarcinoma patients and should be further explored as a decision support tool for their treatment.

Abstract A22: An LKB1-CRTC1 circuit regulates glycosylated COX-2 and predicts drug response in lung cancer

Cyclooxygenase-2 (COX-2) directs the synthesis of prostaglandins important for mitogenic signaling. Here we report that COX-2 is a transcriptional target of the CREB co-activator CRTC1. In addition, we detected a correlation between the LKB1-null status and presence of 72/74 kDa glycosylated COX-2, but not inactive hypoglycosylated COX-2 in fresh lung adenocarcinoma samples. Since CRTC1 is suppressed by cytoplasmic shuttling following LKB1/AMPK/SIK phosphorylation, we developed an LKB1 signature in lung cancer to search the Connectivity-MAP drug response database. Remarkably, all high-ranking drugs positively associated with the LKB1-null signature were known CRTC1 activators. Somatic LKB1 mutations are present in 20% of lung adenocarcinomas and we observed growth and cell motility inhibition with COX-2 inhibitors in LKB1-null lung cancer cells with activated CRTC1, but negligible inhibition in LKB1-wildtype cells. In summary, the CREB co-activator CRTC family directly links LKB1 with COX-2 activation and provides a new framework for selecting patients for COX-2 inhibition. Citation Format: Chunxia Cao, Ruli Gao, Min Zhang, Antonio L. Amelio, Mohammad Fallahi, Zirong Chen, Yumei Gu, Chengbin Hu, Eric A. Welsh, Brienne E. Engel, Eric Haura, W. Douglas Cress, Lizi Wu, Maria Zajac-Kaye, Frederic J. Kaye. An LKB1-CRTC1 circuit regulates glycosylated COX-2 and predicts drug response in lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A22.

The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts

We previously characterized the retinoblastoma tumor suppressor protein (Rb) as a regulator of adherens junction assembly and cell-to-cell adhesion in osteoblasts. This is a novel function since Rb is predominantly known as a cell cycle repressor. Herein, we characterized the molecular mechanisms by which Rb performs this function, hypothesizing that Rb controls the activity of known regulators of adherens junction assembly. We found that Rb represses the expression of the p21-activated protein kinase (Pak1), an effector of the small Rho GTPase Rac1. Rac1 is a well-known regulator of adherens junction assembly whose increased activity in cancer is linked to perturbations of intercellular adhesion. Using nuclear run-on and luciferase reporter transcription assays, we found that Pak1 repression by Rb is transcriptional, without affecting Pak1 mRNA and protein stability. Pak1 promoter bioinformatics showed multiple E2F1 binding sites within 155 base pairs of the transcriptional start site, and a Pak1-promoter region containing these E2F sites is susceptible to transcriptional inhibition by Rb. Chromatin immunoprecipitations showed that an Rb-E2F complex binds to the region of the Pak1 promoter containing the E2F1 binding sites, suggesting that Pak1 is an E2F target and that the repressive effect of Rb on Pak1 involves blocking the trans-activating capacity of E2F. A bioinformatics analysis showed elevated Pak1 expression in several solid tumors relative to adjacent normal tissue, with both Pak1 and E2F increased relative to normal tissue in breast cancer, supporting a cancer etiology for Pak1 up-regulation. Therefore, we propose that by repressing Pak1 expression, Rb prevents Rac1 hyperactivity usually associated with cancer and related to cytoskeletal derangements that disrupt cell adhesion, consequently enhancing cancer cell migratory capacity. This de-regulation of cell adhesion due to Rb loss could be part of the molecular events associated with cancer progression and metastasis.

Abstract 4159: Characterization of three recurring STK11/LKB1 mutants in lung adenocarcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The STK11 gene encodes the tumor suppressor serine/threonine protein kinase 11 also known as liver kinase β1 (LKB1). The STK11 gene product regulates cellular energy metabolism and cell polarity by activating AMP-activated protein kinase (AMPK) and other members of the AMPK family. Although rare in most human cancers, mutations in the STK11 gene are third most frequent in lung adenocarcinoma after KRAS and TP53. Mutational status for STK11, EGFR, and KRAS was determined in tumor DNA from 468 lung adenocarcinoma patients and STK11 gene expression measured on an Affymetrix GeneChip microarray. Immunohistochemistry (IHC) was performed on a subset of the patients (N = 140). Survival analyses were performed using Kaplan-Meier survival curves and Cox proportional hazard regression. Three recurring mutations (D194Y, P281fs*6, F354L) were evaluated via Western Blot for AMPK phosphorylation. These mutants were also evaluated via immunoprecipitation and Western Blot for binding to two accessory subunits (MO25 and STRAD) that, along with STK11, make up the active trimeric complex. STK11 was mutated in 17.5% of all tumors evaluated with 66 different STK11 mutations noted, only 8 of which occurred more than once. STK11 mutant samples had lower levels of STK11 protein as measured by IHC (< 2+: 50.0% mutant vs. 17.9% WT, P < 0.001). Among three STK11 mutants that were characterized, D194Y and F354L were both able to bind MO25 and STRAD, but these interactions were lost in P281fs*6. The P281fs*6 mutant lacks kinase activity while the D194Y mutant has dominant negative activity, suppressing AMPK activation by WT protein. P281fs*6 mutant tumors had lower gene expression compared to other mutants while WT tumors had the highest expression. D194Y mutant tumors had expression levels similar to WT tumors, higher than the average mutant expression. STK11 gene expression for F354L tumors was higher than the mutant tumor average, but lower than WT. The F354L variant is able to phosphorylate AMPK indicating a potential single nucleotide polymorphism (SNP) supported by its appearance in the 1000 Genomes Project. The F354L variant is more prevalent in lung adenocarcinoma patients compared to the general population which may suggest that it is a genetic susceptibility factor. Interestingly, when all STK11 mutant samples were combined and compared to WT tumors, STK11 mutant tumors were significantly associated with a reduced risk of death (HR = 0.28; 95% CI 0.10 - 0.78) among stage I and II patients adjusted for prognostic factors. This study revealed that STK11 mutant tumors as a whole had lower levels of STK11 protein as measured by IHC, and were associated with a significantly reduced risk of death among early stage patients with lung adenocarcinoma. Mutation status and protein levels alone cannot be used to make treatment decisions, however, as the three recurring mutants characterized caused three disparate effects on the STK11 pathway. Citation Format: Brienne E. Engel, Matthew B. Schabath, Zachary J. Thompson, Steven A. Eschrich, Stephen G. Brantley, Anastasia R. Belock, Anders Berglund, Jhanelle E. Gray, Amer A. Beg, Eric B. Haura, W. Douglas Cress. Characterization of three recurring STK11/LKB1 mutants in lung adenocarcinoma. [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 4159. doi:10.1158/1538-7445.AM2014-4159

Abstract C19: Molecular biology of lung cancer in Puerto Ricans

Lung cancer is a leading cancer killer, lung adenocarcinoma (AC) being its most common type. Several studies suggest that the frequency or nature of genetic events driving AC differ among ethnic populations. These differences impact treatment decisions as targeted agents are under development for a number of genes that drive lung cancer. For example, mutations in the epidermal growth factor receptor (EGFR) represent a driving event in a substantial number of AC patients that can be targeted with tyrosine kinase inhibitors. With the funding of a U54 Partnership grant between the Ponce School of Medicine in Ponce, PR and the Moffitt Cancer Center in Tampa, Florida we are constructing a molecular database for Puerto Rican Lung Cancer patients. Sample collection includes acquisition of fresh frozen and paraffin-embedded tissues via the U54-funded Puerto Rico tissue bank. DNA analysis of these tissues includes ancestry markers and exome sequencing of genes commonly altered in AC, including KRAS, TP53, EGFR, STK11, CDKN1C and RB1. Initial results suggest that the frequency of KRAS mutations in this population are significantly lower than those seen in a white mainland cohort, whereas the rate of EGFR and STK11 mutations is elevated. Since both EFGR and STK11 are potentially targetable mutations these data suggest that Puerto Rican AC patients would greatly benefit from genetic screening. Our U54 project also probes the role of the retinoblastoma protein (Rb) in lung cancer. Rb was the first tumor suppressor to be discovered, and yet its potency as a tumor suppressor remains only partially explained. We are interested in characterizing how Rb loss contributes to the aggressiveness of small cell lung carcinomas (SCLC). SCLC are poorly differentiated tumors with early proclivity for metastasis. Prognosis is poor with only 10-20% of patients achieving long-term disease-free intervals. Still, SCLC aggressivity remains to be molecularly explained. Interestingly, SCLC show high rates of Rb inactivation, which led us to hypothesize that in SCLC, Rb loss during early tumorigenesis facilitates both over-proliferation and early metastasis. Supporting our hypothesis, we found that the Rb-deficient SCLC cell line H187 lacks expression of E-cadherin while overexpressing N-cadherin, relative to Rb-expressing non-small cell lung carcinoma cell lines H1666 and H1650. We confirmed the Rb-positive status of H1666 and H1650 and the Rb-negative status of H187 by immunoblot. These results suggest that Rb loss in lung cancer cell lines provokes a cadherin switch indicative of an epithelial-to-mesenchymal transition. Consistently, we found overexpression of the mesenchymal marker vimentin in H187 relative to H1666 and H1650. When compared to H1666 and H1650, H187 lacked expression of the adherens junction component α-catenin and of the cytoskeletal adapter Eplin. Furthermore, H187 cells show up-regulated levels of Pak1, a Rac1 and Cdc42 effector whose up-regulation is associated to adherens junction disruption. Tiam-1, a Rac1 and Cdc42 activator, is also up-regulated in H187 relative to H1666 and H1650. Together, our data in lung cell lines suggests that Rb loss results in adherens junction alterations possibly due to transcriptional alteration of cell adhesion genes, aberrant regulation of the Rho GTPases Rac1 and Cdc42, and disruption of the cortical cytoskeleton. Based on our data, we propose that Rb loss in SCLC, not only leads to an increased proliferative capacity during early tumorigenesis, but also exacerbates early tumor aggressiveness by perturbing cellular adhesion, which facilitates tumor cell detachment and metastasis. Our ultimate goal is to link the genetic alterations we found in Puerto Ricans to Rb function, cell adhesion, and tumor aggressivity. Citation Format: Pedro G. Santiago-Cardona, Brienne E. Engel, Jonathan Gonzalez-Flores, Ruth Cruz-Cosme, Teresita Munoz-Antonia, W. Douglas Cress. Molecular biology of lung cancer in Puerto Ricans. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C19. doi:10.1158/1538-7755.DISP13-C19