Somatic mutations in EGFR, the gene encoding the epidermal growth factor receptor, have been found in the tumors of 10-40% of patients with non-small-cell lung cancer (NSCLC). These mutations activate the EGFR tyrosine kinase and are associated with adenocarcinoma histology, female gender and a non-smoking history. Lung cancer patients with EGFR mutations frequently experience rapid and sustained shrinkage of primary and metastatic disease after treatment with the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. In a phase III trial, treatment with erlotinib conferred a survival benefit, but the proportion of patients who experienced this benefit exceeded the expected frequency of EGFR mutations. In addition, a small proportion of patients whose tumors shrank in response to EGFR TKIs had no evidence of EGFR mutations. Together, these findings suggest that factors other than EGFR mutations confer sensitivity to EGFR inhibition.
EGFR forms homodimers and heterodimers with the other ErbB family members ErbB2, ErbB3 and ErbB4. These dimeric complexes have distinct ligand binding and signaling activities. For example, ErbB3 is unique in that it lacks a functional kinase domain. Despite this deficiency, ErbB3 undergoes trans-phosphorylation in complex with other ErbBs and activates downstream kinases such as phosphatidylinositol 3-kinase (PI3K) in response to ligand binding. Recent studies have implicated ErbB3 in the sensitivity of NSCLC cell lines to EGFR inhibition. Together, these findings suggest that aberrant expression of ErbB family members contributes to the responsiveness of NSCLC cells to EGFR inhibition.
A recent report linked de novo resistance to treatment with EGFR TKIs in lung adenocarcinoma patients with somatic mutations in KRAS. A growing body of evidence indicates that KRAS mutations are important in the development of lung adenocarcinoma. They occur in 30-50% of lung adenocarcinomas and are mutually exclusive from mutations in EGFR. Mice that express mutant Kras develop lung adenocarcinoma rapidly and with high penetrance. In this study, we investigated the role of KRAS mutations in the resistance of lung adenocarcinoma to EGFR TKIs. We examined KrasLA1 mice, which develop lung adenocarcinoma through somatic activation of a Kras allele carrying an activating mutation in codon 12 (G12D). Alveolar epithelial cells in this mouse model recapitulate the series of morphologic stages through which human atypical alveolar hyperplasia (AAH) evolves into adenocarcinoma. We found that the presence of KRAS mutations is not sufficient to confer resistance to EGFR inhibition and reveal a novel mechanism of response to EGFR inhibition that is potentially relevant to lung cancer patients. Specifically, we found high expression of ErbB family members, ErbB ligands, or both in three models that were sensitive to EGFR inhibition, including alveolar epithelial neoplastic lesions in mice that develop lung adenocarcinoma by oncogenic KRAS, human lung adenocarcinoma cell lines and tumor biopsies from lung adenocarcinoma patients. Thus, lung adenocarcinoma cells that depend on EGFR for survival constitutively activate the receptor through a combination of genetic mutations and over-expression of EGFR dimeric partners and their ligands.
Regulation of phosphoinositides is important to tumorigenesis. PTEN (phosphatase and tensin homologue deleted from chromosome 10) is a dual-specificity phosphatase that dephosphorylates the 3' sites of the phosphoinositides PI(3,4)P2 and PI(3,4,5)P3. Endogenous PI(3,4,5)P3 levels are also regulated by PI3K, which phosphorylates the D3 position of PI on PI(4)P and PI(4,5)P to produce PI(3,4)P2 and PI(3,4,5)P3. PI(3,4,5)P3 and PI(3,4)P2 recruit the pleckstrin homology domains of specific intracellular proteins to the plasma membrane, an essential event in the activation of PI3K-dependent kinases such as phosphoinositide-dependent kinase-1 and protein kinase B/AKT, which have a key role in cellular survival and transformation.
Several genetic events previously described in human lung cancer activate PI3K, including amplification of PI3KCA and activating mutations in PI3KCA, EGFR or KRAS. Several biochemical events have been identified downstream of AKT that enhance cell survival, increase cell proliferation and alter cell metabolism. AKT phosphorylates and inactivates downstream substrates, including BAD, FOXO proteins, GSK3 and tuberin, the protein product of Tsc2. Phosphorylation of tuberin leads to activation of mammalian target of rapamycin (mTOR, encoded by the gene frap1 in mice), a critical mediator of protein translation. mTOR substrates required for protein translation include the serine/threonine kinase S6K1 (p70S6K/p85S6K) and the 4E binding protein 1 (4EBP1). Phosphorylation of 4EBP1 causes it to dissociate from eukaryotic initiation factor (eIF) 4E, which then binds to the eIF4G scaffold protein, promoting the assembly of the eIF4F initiation complex. Of note, eIF4E is over-expressed in BAC and lung adenocarcinoma. These cumulative observations have led to derivatives of the mTOR inhibitor rapamycin being tested in clinical trials of lung cancer.
We have found that mTOR inhibition blocked malignant progression in K-rasLA1 mice. Levels of phosphorylated S6Ser236/235 (p-S6), a downstream mediator of mTOR, increased with malignant progression (normal alveolar epithelial cells to adenocarcinoma) in K-rasLA1 mice and in patients with lung adenocarcinoma. Atypical alveolar hyperplasia (AAH), an early neoplastic change, was prominently associated with macrophages and expressed high levels of p-S6. mTOR inhibition in K-rasLA1 mice by treatment with the rapamycin analog CCI-779 reduced the size and number of early epithelial neoplastic lesions (AAH and adenomas) and induced apoptosis of intra-epithelial macrophages. LKR-13 cells were resistant to treatment with CCI-779 in vitro. However, LKR-13 cells grown as syngeneic tumors recruited macrophages, and those tumors regressed in response to treatment with CCI-779. Lastly, conditioned medium from primary cultures of alveolar macrophages stimulated the proliferation of LKR-13 cells. These findings provide evidence that the expansion of lung adenocarcinoma precursors induced by oncogenic K-ras requires mTOR-dependent signaling and suggest that host factors derived from macrophages may contribute to adenocarcinoma progression.
Ligands for Chemokine Receptors
NSCLC is highly invasive and frequently metastatic at the time of diagnosis. Because metastatic NSCLC is refractory to all known interventions, a logical approach to reducing the mortaility from NSCLC is to intervene prior to the development of invasive disease. Success in this effort will require understanding the genetic and biochemical changes in NSCLC that confer invasive properties.
Biologic features of NSCLC that correlate with advanced stages of disease include the infiltration of neutrophils and enhanced tumor vasculature. The CXC chemokines recruit inflammatory cells and endothelial cells to tumors and are autocrine growth factors for certain types of cancer cells. The CXC chemokine family can be divided into two groups according to the presence or absence of an ELR (Glu-Leu-Arg) motif located immediately before the first cysteine residue at the amino terminus. ELR+ CXC chemokines are known for their potent chemoattraction for neutrophils and to a lesser extent T cells as well as their ability to promote angiogenesis (9-11). There are three known chemokine receptors, CXCR1, CXCR2 and Duffy antigen receptor for chemokines, which are G-protein-coupled receptors. CXCR2 is expressed on endothelial cells, thereby promoting angiogenesis in tumors that express CXC chemokines.
CXCL8 (also known as interleukin-8), a ligand for CXCR2, is present in freshly isolated specimens of human NSCLC and has been implicated as the dominant mediator of aberrant angiogenesis in a syngeneic murine Lewis lung cancer model and in human NSCLC/SCID mouse chimera. A recent study demonstrated that CXCL8 is a transcriptional target of Ras signaling and is required for the initiation of tumor-associated inflammation and neovascularization in xenograft models. Mutations in the proto-oncogene KRAS occur in 30-50% of lung adenocarcinomas, the most common sub-type of NSCLC, and expression of mutant KRAS in the alveolar epithelium leads to the development of lung adenocarcinoma in mice. In addition to its role in the transformation of alveolar epithelial cells, the presence of KRAS mutations is a predictor of shorter survival in NSCLC patients. Thus, studies are warranted to investigate the role of CXCL8 in lung adenocarcinoma induced by mutant KRAS.
We have investigated the role of the murine homologues of CXCL8, KC and MIP-2, in KrasLA1 mice. Malignant progression (normal alveolar epithelial cells to adenocarcinoma) in KrasLA1 mice was associated with enhanced intra-lesional vascularity and inflammation, which are hallmarks of chemoattraction by CXCR2 ligands. CXCR2, KC and MIP-2 were highly expressed in alveolar lesions in KrasLA1 mice and in vitro in alveolar inflammatory cells and LKR-13 cells. Treatment of KrasLA1 mice with a neutralizing antibody against CXCR2 decreased the size and number of early lung lesions, blocked the progression of epithelial hyperplasia to adenoma and induced apoptosis of vascular endothelial cells within alveolar lesions. Whereas the proliferation of LKR-13 cells in vitro was resistant to treatment with the CXCR2 neutralizing antibody, LKR-13 cells established as syngeneic tumors were sensitive to treatment, supporting a role for the tumor microenvironment in CXCR2 actions. CXCR2 was required for the migration of murine endothelial cells, alveolar inflammatory cells and LKR-13 cells in vitro. Thus, high expression of CXCR2 ligands may contribute to the expansion of early alveolar neoplastic lesions induced by oncogenic KRAS.
Interactions between PI3K and Stress Kinase Signaling Through NFkB
While PTEN gene expression is frequently silenced in lung cancer, PTEN genetic deletion is a rare event. Of note, PTEN expression is transcriptionally suppressed by tumor necrosis factor-a (TNF) through NFkB, a heterodimeric transcription factor that is constitutively activated in lung cancer. NFkB consists of the transactivation subunit RelA/p65 and the DNA-binding subunits p50 (NFkB1) and p52 (NFkB2), which are processed from the precursors p105 and p100, respectively. In unstimulated conditions, NFkB is sequestered in the cytoplasm by inhibitor of NFkB (IkB) and remains transcriptionally inactive. Upon stimulation by inflammatory cytokines or peptide growth factors, IkB is phosphorylated by IkB kinase (IKK) and undergoes proteasome-dependent degradation. The released NFkB translocates into the nucleus and regulates the expression of target genes with key roles in the prevention of apoptosis, promotion of tumor growth and activation of inflammatory responses.
Lung cancer cells undergo apoptosis in response to PI3K pathway inhibition. We previously found that a stress kinase, mitogen-activated protein kinase kinase-4 (MKK4), activates pro-survival signals in lung cancer cells and can rescue them from the pro-apoptotic effect of PI3K inhibition. MKK4 is a dual-specificity kinase that is activated by environmental stresses, including exposure of cells to UV irradiation, DNA damage, growth factors or inflammatory cytokines. Consistent with a pro-survival role, disruption of MKK4 causes embryonic death and increases liver cell apoptosis. However, the mechanisms by which MKK4 regulates cell survival have not been fully defined.
We hypothesized that MKK4 promotes cell survival by potentiating PI3K-dependent signaling, which we addressed by using MKK4-null and -wild-type mouse embryo fibroblasts (MEFs). We showed in MEFs and lung cancer cells that the absence of MKK4 was associated with enhanced susceptibility to apoptosis, increased expression of PTEN and a corresponding reduction in phosphoinositides. In MKK4-null MEFs, transcriptional suppression of PTEN gene expression by NFkB was diminished, NFkB and its upstream activator IKKa were unresponsive to tumor necrosis factor-a and processing of NFkB precursors (p105 and p100) to p50 (NFkB1) and p52 (NFkB2) was reduced. Thus, MKK4 is required for the maturation of NFkB precursors and promotes cell survival by inhibiting PTEN gene expression through NFkB activation.