Faculty Bibliography

Cancer develops through genetic and epigenetic alterations that allow unrestrained proliferation and increased survival. Using a genetic RNAi screen, we previously identified hundreds of suppressors of tumorigenesis and/or proliferation (STOP) genes that restrain normal cell proliferation. Our STOP gene set was significantly enriched for known and putative tumor suppressor genes. Here, we report a tumor-suppressive role for one STOP gene, phosphatase and actin regulator 4 (PHACTR4). Phactr4 is one of four members of the largely uncharacterized Phactr family of protein phosphatase 1 (PP1)-and actin-binding proteins. Our work suggests that Phactr4 restrains normal cell proliferation and transformation. Depletion of Phactr4 with multiple shRNAs leads to increased proliferation and soft agar colony formation. Phactr4 acts, in part, through an Rb-dependent pathway, because Rb phosphorylation is maintained upon growth factor withdrawal in Phactr4-depleted cells. Examination of tumor copy number analysis and sequencing revealed that PHACTR4 is significantly deleted and mutant in many tumor subtypes. Furthermore,cancer cell lines with reduced Phactr4 expression exhibit tumor suppressor hypersensitivity upon Phactr4 complementation,leading to reduced proliferation, transformation, and tumor formation. Thus, Phactr4 acts as a tumor suppressor that is deleted and mutant in several cancers.

The clinical efficacy of EGF receptor (EGFR) kinase inhibitors gefitinib and erlotinib is limited by the development of drug resistance. The most common mechanism of drug resistance is the secondary EGFR T790M mutation. Strategies to overcome EGFR T790M-mediated drug resistance include the use of mutant selective EGFR inhibitors, including WZ4002, or the use of high concentrations of irreversible quinazoline EGFR inhibitors such as PF299804. In the current study, we develop drug-resistant versions of the EGFR-mutant PC9 cell line, which reproducibly develops EGFR T790M as a mechanism of drug resistance to gefitinib. Neither PF299804-resistant nor WZ4002-resistant clones of PC9 harbor EGFR T790M. Instead, they have shown activated insulin-like growth factor receptor (IGF1R) signaling as a result of loss of expression of IGFBP3 with the IGF1R inhibitor, BMS 536924, restoring EGFR inhibitor sensitivity. Intriguingly, prolonged exposure to either PF299804 or WZ4002 results in the emergence of a more drug-resistant subclone that exhibits ERK activation. A MEK inhibitor, CI-1040, partially restores sensitivity to the EGFR/IGF1R inhibitor combination. Moreover, an IGF1R or MEK inhibitor used in combination with either PF299804 or WZ4002 completely prevents the emergence of drug-resistant clones in this model system. Our studies suggest that more effective means of inhibiting EGFR T790M will prevent the emergence of this common drug resistance mechanism in EGFR-mutant non-small cell lung cancer. However, multiple drug resistance mechanisms can still emerge. Preventing the emergence of drug resistance, by targeting pathways that become activated in resistant cancers, may be a more effective clinical strategy.

KRAS is the most commonly mutated oncogene, yet no effective targeted therapies exist for KRAS mutant cancers. We developed a pooled shRNA-drug screen strategy to identify genes that, when inhibited, cooperate with MEK inhibitors to effectively treat KRAS mutant cancer cells. The anti-apoptotic BH3 family gene BCL-XL emerged as a top hit through this approach. ABT-263 (navitoclax), a chemical inhibitor that blocks the ability of BCL-XL to bind and inhibit pro-apoptotic proteins, in combination with a MEK inhibitor led to dramatic apoptosis in many KRAS mutant cell lines from different tissue types. This combination caused marked in vivo tumor regressions in KRAS mutant xenografts and in a genetically engineered KRAS-driven lung cancer mouse model, supporting combined BCL-XL/MEK inhibition as a potential therapeutic approach for KRAS mutant cancers.

Interleukin-6 (IL-6) is involved in lung cancer tumorigenesis, tumor progression, metastasis, and drug resistance. Previous studies show that blockade of IL-6 signaling can inhibit tumor growth and increase drug sensitivity in mouse models. Clinical trials in non-small cell lung cancer (NSCLC) reveal that IL-6 targeted therapy relieves NSCLC-related anemia and cachexia, although other clinical effects require further study. We crossed IL-6(-/-) mice with Kras(G12D) mutant mice, which develop lung tumors after activation of mutant Kras(G12D), to investigate whether IL-6 inhibition contributes to tumor progression and survival time in vivo. Kras(G12D); IL-6(-/-) mice exhibited increased tumorigenesis, but slower tumor growth and longer survival, than Kras(G12D) mice. Further, in order to investigate whether IL-6 deletion contributes to suppression of lung cancer metastasis, we generated Kras(G12D); p53(flox/flox); IL-6(-/-) mice, which developed lung cancer with a trend for reduced metastases and longer survival than Kras(G12D); p53(flox/flox) mice. Tumors from Kras(G12D); IL-6(-/-) mice showed increased expression of TNFalpha and decreased expression of CCL-19, CCL-20 and phosphorylated STAT3(pSTAT3) than Kras(G12D) mice; however, these changes were not present between tumors from Kras(G12D); p53(flox/flox); IL-6(-/-) and Kras(G12D); p53(flox/flox) mice. Upregulation of pSTAT3 and phosphorylated AKT(pAKT) were observed in Kras(G12D) tumors with p53 deletion. Taken together, these results indicate that IL-6 deletion accelerates tumorigenesis but delays tumor progression and prolongs survival time in a Kras-driven mouse model of lung cancer. However, these effects can be attenuated by p53 deletion.

Germline mutations in the LKB1 gene (also known as STK11) cause the Peutz-Jeghers Syndrome, and somatic loss of LKB1 has emerged as causal event in a wide range of human malignancies, including melanoma, lung cancer, and cervical cancer. The LKB1 protein is a serine-threonine kinase that phosphorylates AMP-activated protein kinase (AMPK) and other downstream targets. Conditional knockout studies in mouse models have consistently shown that LKB1 loss promotes a highly-metastatic phenotype in diverse tissues, and human studies have demonstrated a strong association between LKB1 inactivation and tumor recurrence. Furthermore, LKB1 deficiency confers sensitivity to distinct classes of anticancer drugs. The ability to reliably identify LKB1-deficient tumors is thus likely to have important prognostic and predictive implications. Previous research studies have employed polyclonal antibodies with limited success, and there is no widely-employed immunohistochemical assay for LKB1. Here we report an assay based on a rabbit monoclonal antibody that can reliably detect endogenous LKB1 protein (and its absence) in mouse and human formalin-fixed, paraffin-embedded tissues. LKB1 protein levels determined through this assay correlated strongly with AMPK phosphorylation both in mouse and human tumors, and with mRNA levels in human tumors. Our studies fully validate this immunohistochemical assay for LKB1 in paraffin-embedded formalin tissue sections. This assay should be broadly useful for research studies employing mouse models and also for the development of human tissue-based assays for LKB1 in diverse clinical settings.

Despite the ongoing "war on cancer," cancer remains one of the major causes of human morbidity and mortality. A new paradigm of targeted therapies holds the most promise for the future, making identification of tumor-specific therapeutic targets of prime importance. ERBB2/HER2, best known for its role in breast cancer tumorigenesis, can be targeted by two types of pharmacological manipulation: antibody therapy against the extracellular receptor domain and small molecule compounds against the intracellular tyrosine kinase domain. Aberrant activation of ERBB2 by gene amplification has been shown to participate in the pathophysiology of breast, ovarian, gastric, colorectal, lung, brain, and head and neck tumors. However, the advent of next-generation sequencing technologies has enabled efficient identification of activating molecular alterations of ERBB2. In this review, we will focus on the functional role of these somatic mutations that cause ERBB2 receptor activation. We will additionally discuss the current preclinical and clinical therapeutic strategies for targeting mutationally activated ERBB2.

Somatic mutation of the tumor suppressor gene LKB1 occurs frequently in lung cancer where it causes tumor progression and metastasis, but the underlying mechanisms remain mainly unknown. Here, we show that the oncogene NEDD9 is an important downstream mediator of lung cancer progression evoked by LKB1 loss. In de novo mouse models, RNAi-mediated silencing of Nedd9 inhibited lung tumor progression, whereas ectopic NEDD9 expression accelerated this process. Mechanistically, LKB1 negatively regulated NEDD9 transcription by promoting cytosolic translocation of CRTC1 from the nucleus. Notably, ectopic expression of either NEDD9 or CRTC1 partially reversed the inhibitory function of LKB1 on metastasis of lung cancer cells. In clinical specimens, elevated expression of NEDD9 was associated with malignant progression and metastasis. Collectively, our results decipher the mechanism through which LKB1 deficiency promotes lung cancer progression and metastasis, and provide a mechanistic rationale for therapeutic attack of these processes.

Patients with lung cancer with activating mutations in the EGF receptor (EGFR) kinase, who are treated long-term with tyrosine kinase inhibitors (TKI), often develop secondary mutations in EGFR associated with resistance. Mice engineered to develop lung adenocarcinomas driven by the human EGFR T790M resistance mutation are similarly resistant to the EGFR TKI erlotinib. By tumor volume endpoint analysis, these mouse tumors respond to BIBW 2992 (an irreversible EGFR/HER2 TKI) and rapamycin combination therapy. To correlate EGFR-driven changes in the lung with response to drug treatment, we conducted an integrative analysis of global transcriptome and metabolite profiling compared with quantitative imaging and histopathology at several time points during tumor progression and treatment. Responses to single-drug treatments were temporary, whereas combination therapy elicited a sustained response. During tumor development, metabolomic signatures indicated a shift to high anabolic activity and suppression of antitumor programs with 11 metabolites consistently present in both lung tissue and blood. Combination drug treatment reversed many of the molecular changes found in tumored lung. Data integration linking cancer signaling networks with metabolic activity identified key pathways such as glutamine and glutathione metabolism that signified response to single or dual treatments. Results from combination drug treatment suggest that metabolic transcriptional control through C-MYC and SREBP, as well as ELK1, NRF1, and NRF2, depends on both EGFR and mTORC1 signaling. Our findings establish the importance of kinetic therapeutic studies in preclinical assessment and provide in vivo evidence that TKI-mediated antiproliferative effects also manifest in specific metabolic regulation.