Person:

Wong, Kwok-Kin

Animal models which allow the temporal regulation of gene activities are valuable for dissecting gene function in tumorigenesis. Here we have constructed a conditional inducible estrogen receptor-K-ras(^{G12D}) (ER-K-ras(^{G12D})) knock-in mice allele that allows us to temporally switch on or off the activity of K-ras oncogenic mutant through tamoxifen administration. In vitro studies using mice embryonic fibroblast (MEF) showed that a dose of tamoxifen at 0.05 (\mu)M works optimally for activation of ER-K-ras(^{G12D}) independent of the gender status. Furthermore, tamoxifen-inducible activation of K-ras(^{G12D}) promotes cell proliferation, anchor-independent growth, transformation as well as invasion, potentially via activation of downstream MAPK pathway and cell cycle progression. Continuous activation of K-ras(^{G12D}) in vivo by tamoxifen treatment is sufficient to drive the neoplastic transformation of normal lung epithelial cells in mice. Tamoxifen withdrawal after the tumor formation results in apoptosis and tumor regression in mouse lungs. Taken together, these data have convincingly demonstrated that K-ras mutant is essential for neoplastic transformation and this animal model may provide an ideal platform for further detailed characterization of the role of K-ras oncogenic mutant during different stages of lung tumorigenesis.

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.

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.

Oncogene–induced DNA damage elicits genomic instability in epithelial cancer cells, but apoptosis is blocked through inactivation of the tumor suppressor p53. In hematological cancers, the relevance of ongoing DNA damage and mechanisms by which apoptosis is suppressed are largely unknown. We found pervasive DNA damage in hematologic malignancies including multiple myeloma, lymphoma and leukemia, which leads to activation of a p53–independent, pro-apoptotic network centered on nuclear relocalization of ABL1 kinase. Although nuclear ABL1 triggers cell death through its interaction with the Hippo pathway co–activator YAP1 in normal cells, we show that low YAP1 levels prevent nuclear ABL1–induced apoptosis in these hematologic malignancies. YAP1 is under the control of a serine–threonine kinase, STK4. Importantly, genetic inactivation of STK4 restores YAP1 levels, triggering cell death in vitro and in vivo. Our data therefore identify a novel synthetic–lethal strategy to selectively target cancer cells presenting with endogenous DNA damage and low YAP1 levels.

Non-small cell lung cancers (NSCLCs) that harbor an oncogenic KRAS mutation are often associated with resistance to targeted therapies. The MUC1-C transmembrane protein is aberrantly overexpressed in NSCLCs and confers a poor outcome; however, the functional role for MUC1-C in mutant KRAS NSCLC cells has remained unclear. The present studies demonstrate that silencing MUC1-C in A549/KRAS(G12S) and H460/KRAS(Q61H) NSCLC cells is associated with downregulation of AKT signaling and inhibition of growth. Overexpression of a MUC1-C(CQC→AQA) mutant, which inhibits MUC1-C homodimerization and function, suppressed both AKT and MEK activation. Moreover, treatment with GO-203, an inhibitor of MUC1-C homodimerization, blocked AKT and MEK signaling and decreased cell survival. The results further demonstrate that targeting MUC1-C suppresses expression of the ZEB1 transcriptional repressor by an AKT-mediated mechanism, and in turn induces miR-200c. In concert with these effects on the ZEB1/miR-200c regulatory loop, targeting MUC1-C was associated with reversal of the epithelial-mesenchymal transition (EMT) and inhibition of self-renewal capacity. Loss of MUC1-C function also attenuated KRAS independence and inhibited growth of KRAS mutant NSCLC cells as tumors in mice. These findings support a model in which targeting MUC1-C inhibits mutant KRAS signaling in NSCLC cells and thereby reverses the EMT phenotype and decreases self-renewal.

Lineage transition in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) of non-small cell lung cancer, as implicated by clinical observation of mixed ADC and SCC pathologies in adenosquamous cell carcinoma, remains a fundamental yet unsolved question. Here we provide in vivo evidence showing the transdifferentiation of lung cancer from ADC to SCC in mice: Lkb1-deficient lung ADC progressively transdifferentiates into SCC, via a pathologically mixed mAd-SCC intermediate. We find that reduction of lysyl oxidase (Lox) in Lkb1-deficient lung ADC decreases collagen disposition and triggers extracellular matrix remodelling and upregulates p63 expression, a SCC lineage survival oncogene. Pharmacological Lox inhibition promotes the transdifferentiation, whereas ectopic Lox expression significantly inhibits this process. Notably, ADC and SCC show differential responses to Lox inhibition. Collectively, our findings demonstrate the de novo transdifferentiation of lung ADC to SCC in mice and provide mechanistic insight that may have important implications for lung cancer treatment.

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 KrasG12D mutant mice, which develop lung tumors after activation of mutant KrasG12D, to investigate whether IL-6 inhibition contributes to tumor progression and survival time in vivo. KrasG12D; IL-6-/- mice exhibited increased tumorigenesis, but slower tumor growth and longer survival, than KrasG12D mice. Further, in order to investigate whether IL-6 deletion contributes to suppression of lung cancer metastasis, we generated KrasG12D; p53flox/flox; IL-6-/- mice, which developed lung cancer with a trend for reduced metastases and longer survival than KrasG12D; p53flox/flox mice. Tumors from KrasG12D; IL-6-/- mice showed increased expression of TNFα and decreased expression of CCL-19, CCL-20 and phosphorylated STAT3 (pSTAT3) than KrasG12D mice; however, these changes were not present between tumors from KrasG12D; p53flox/flox; IL-6-/- and KrasG12D; p53flox/flox mice. Upregulation of pSTAT3 and phosphorylated AKT (pAKT) were observed in KrasG12D 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.

Cancer stem cells (CSCs) are considered one of the key contributors to chemoresistance and tumor recurrence. Therefore, the precise identification of reliable CSC markers and clarification of the intracellular signaling involved in CSCs remains a great challenge in fields relating to cancer biology. Here, we implemented a novel chemoresistant prostate cancer patient-derived xenograft (PDX) model in NOD/SCID mice and identified CD54 as a candidate gene among the most highly enriched gene expression profiles in prostate tumors exposed to chronic cisplatin administration. Additional in vitro and in vivo assays showed that CD54 played a critical role in the self-renewal and tumorigenesis of prostate CSCs. Moreover, silencing CD54 greatly reduced the tumorigenesis of prostate cancers both in vitro and in vivo and significantly extended the survival time of tumor-bearing mice in a prostate cancer xenograft model. Dissection of the molecular mechanism revealed that the p38-Notch1 axis was the main downstream signaling pathway in CD54-mediated regulation of CSCs in prostate cancers. Together, these results established that CD54 could be a novel reliable prostate CSC marker and provided a new potential therapeutic target in prostate cancer via CD54-Notch1 signaling.

Despite compelling antitumour activity of antibodies targeting the programmed death 1 (PD-1): programmed death ligand 1 (PD-L1) immune checkpoint in lung cancer, resistance to these therapies has increasingly been observed. In this study, to elucidate mechanisms of adaptive resistance, we analyse the tumour immune microenvironment in the context of anti-PD-1 therapy in two fully immunocompetent mouse models of lung adenocarcinoma. In tumours progressing following response to anti-PD-1 therapy, we observe upregulation of alternative immune checkpoints, notably T-cell immunoglobulin mucin-3 (TIM-3), in PD-1 antibody bound T cells and demonstrate a survival advantage with addition of a TIM-3 blocking antibody following failure of PD-1 blockade. Two patients who developed adaptive resistance to anti-PD-1 treatment also show a similar TIM-3 upregulation in blocking antibody-bound T cells at treatment failure. These data suggest that upregulation of TIM-3 and other immune checkpoints may be targetable biomarkers associated with adaptive resistance to PD-1 blockade.

The success in lung cancer therapy with Programmed Death (PD)-1 blockade suggests that immune escape mechanisms contribute to lung tumor pathogenesis. We identified a correlation between Epidermal Growth Factor Receptor (EGFR) pathway activation and a signature of immunosuppression manifested by upregulation of PD-1, PD-L1, cytotoxic T lymphocyte antigen-4 (CTLA-4), and multiple tumor-promoting inflammatory cytokines. We observed decreased cytotoxic T cells and increased markers of T cell exhaustion in mouse models of EGFR-driven lung cancer. PD-1 antibody blockade improved the survival of mice with EGFR-driven adenocarcinomas by enhancing effector T cell function and lowering the levels of tumor-promoting cytokines. Expression of mutant EGFR in bronchial epithelial cells induced PD-L1, and PD-L1 expression was reduced by EGFR inhibitors in non-small cell lung cancer cell lines with activated EGFR. These data suggest that oncogenic EGFR signaling remodels the tumor microenvironment to trigger immune escape, and mechanistically link treatment response to PD-1 inhibition.