KRAS mutation status is associated with enhanced dependency on folate metabolism pathways in non-small cell lung cancer cells.

KRAS gene mutation is linked to poor prognosis and resistance to therapeutics in non-small cell lung cancer (NSCLC). In this study, we have explored the possibility of exploiting inherent differences in KRAS-mutant cell metabolism for treatment. This study identified a greater dependency on folate metabolism pathways in KRAS mutant compared with KRAS wild-type NSCLC cell lines. Microarray gene expression and biologic pathway analysis identified higher expression of folate metabolism- and purine synthesis-related pathways in KRAS-mutant NSCLC cells compared with wild-type counterparts. Moreover, pathway analysis and knockdown studies suggest a role for MYC transcriptional activity in the expression of these pathways in KRAS-mutant NSCLC cells. Furthermore, KRAS knockdown and overexpression studies demonstrated the ability of KRAS to regulate expression of genes that comprise folate metabolism pathways. Proliferation studies demonstrated higher responsiveness to methotrexate, pemetrexed, and other antifolates in KRAS-mutant NSCLC cells. Surprisingly, KRAS gene expression is downregulated in KRAS wild-type and KRAS-mutant cells by antifolates, which may also contribute to higher efficacy of antifolates in KRAS-mutant NSCLC cells. In vivo analysis of multiple tumorgraft models in nude mice identified a KRAS-mutant tumor among the pemetrexed-responsive tumors and also demonstrated an association between expression of the folate pathway gene, methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), and antifolate activity. Collectively, we identify altered regulation of folate metabolism in KRAS-mutant NSCLC cells that may account for higher antifolate activity in this subtype of NSCLC.

Truncated ErbB2 expressed in tumor cell nuclei contributes to acquired therapeutic resistance to ErbB2 kinase inhibitors.

ErbB2 tyrosine kinase inhibitors (TKI) block tyrosine autophosphorylation and activation of the full-length transmembrane ErbB2 receptor (p185(ErbB2)). In addition to p185(ErbB2), truncated forms of ErbB2 exist in breast cancer cell lines and clinical tumors. The contribution of these truncated forms, specifically those expressed in tumor cell nuclei, to the development of therapeutic resistance to ErbB2 TKIs has not been previously shown. Here, we show that expression of a 95-kDa tyrosine phosphorylated form of ErbB2, herein referred to as p95L (lapatinib-induced p95) was increased in ErbB2(+) breast cancer cells treated with potent ErbB2 TKIs (lapatinib, GW2974). Expressed in tumor cell nuclei, tyrosine phosphorylation of p95L was resistant to inhibition by ErbB2 TKIs. Furthermore, the expression of p95L was increased in ErbB2(+) breast cancer models of acquired therapeutic resistance to lapatinib that mimic the clinical setting. Pretreatment with proteasome inhibitors blocked p95L induction in response to ErbB2 TKIs, implicating the role of the proteasome in the regulation of p95L expression. In addition, tyrosine phosphorylated C-terminal fragments of ErbB2, generated by alternate initiation of translation and similar in molecular weight to p95L, were expressed in tumor cell nuclei, where they too were resistant to inhibition by ErbB2 TKIs. When expressed in the nuclei of lapatinib-sensitive ErbB2(+) breast cancer cells, truncated ErbB2 rendered cells resistant to lapatinib-induced apoptosis. Elucidating the function of nuclear, truncated forms of ErbB2, and developing therapeutic strategies to block their expression and/or activation may enhance the clinical efficacy of ErbB2 TKIs.

Activation of AMPK is necessary for killing cancer cells and sparing cardiac cells.

ErbB2 targeted therapies represent an attractive strategy in breast cancer. Herceptin, an anti-ErbB2 monoclonal antibody, is an approved treatment for patients with ErbB2-overexpressing breast cancers. ErbB2 signaling can also be blocked using small molecule tyrosine kinase inhibitors, like Lapatinib, that compete with ATP for binding at the ErbB2 catalytic kinase domain. The principal adverse event attributable to Herceptin is cardiac toxicity. Data from clinical trials show that, unlike Herceptin, Lapatinib may have reduced cardiac toxicity. This study was conducted to elucidate pathways which may contribute to cardiac toxicity or survival using Lapatinib and Herceptin. Our results show that treatments directed to ErbB1/2 receptors using GW-2974 (a generic ErbB1/2 inhibitor) activated AMPK, a key regulator in mitochondrial energy production pathways in human cardiac cells and cancer cells. Although Herceptin downregulates tumor survival pathways, AMPK fails to be activated in tumor and cardiac cells. When treated in combination with TNFalpha, a known cytokine associated with cardiac toxicity, GW-2974 protected cardiac cells from cell death whereas Herceptin contributed to TNFalpha-induced cellular killing. Since activity of AMPK in cardiac cells is associated with stress induced survival in response to cytokines or energy depletion, cardiac toxicity by Herceptin may be a consequence of failure to induce stress-related survival mechanisms. Thus, the ability to activate AMPK after treatment with tyrosine kinase inhibitors may be a crucial factor for increased efficacy against the tumor and decreased risk of cardiomyopathy.

A reciprocal tensin-3-cten switch mediates EGF-driven mammary cell migration.

Cell migration driven by the epidermal growth factor receptor (EGFR) propels morphogenesis and involves reorganization of the actin cytoskeleton. Although de novo transcription precedes migration, transcript identity remains largely unknown. Through their actin-binding domains, tensins link the cytoskeleton to integrin-based adhesion sites. Here we report that EGF downregulates tensin-3 expression, and concomitantly upregulates cten, a tensin family member that lacks the actin-binding domain. Knockdown of cten or tensin-3, respectively, impairs or enhances mammary cell migration. Furthermore, cten displaces tensin-3 from the cytoplasmic tail of integrin beta1, thereby instigating actin fibre disassembly. In invasive breast cancer, cten expression correlates not only with high EGFR and HER2, but also with metastasis to lymph nodes. Moreover, treatment of inflammatory breast cancer patients with an EGFR/HER2 dual-specificity kinase inhibitor significantly downregulated cten expression. In conclusion, a transcriptional tensin-3-cten switch may contribute to the metastasis of mammary cancer.

Activation of AMP-activated protein kinase by human EGF receptor 2/EGF receptor tyrosine kinase inhibitor protects cardiac cells.

The human EGF receptor (HER) 2 receptor tyrosine kinase is a survival factor for human cardiomyocytes, and its inhibition may explain the increased incidence of cardiomyopathy associated with the anti-HER2 monoclonal antibody trastuzumab (Genentech, South San Francisco, CA), particularly in patients with prior exposure to cardiotoxic chemotherapies e.g., anthracyclines. Here, we show that GW2974 (HER2/EGF receptor tyrosine kinase inhibitor), but not trastuzumab, activates AMP-activated protein kinase (AMPK), initiating a metabolic stress response in human cardiomyocytes that protects against TNFalpha-induced cell death. GW2974 stimulates calcium dependent fatty acid oxidation in vitro and in the myocardium of GW2974-treated rodents. Calcium chelation or siRNA-targeted AMPK knockdown blocks GW2974 induced fatty acid oxidation. In addition, inhibition of AMPK by a specific inhibitor resulted in increased killing of cardiomyocytes. Elucidating the effects of HER2-targeted therapies on AMPK may predict for risk of cardiomyopathy and provide a novel HER2-targeted strategy designed to protect myocardium from the pro-apoptotic effects of pro-inflammatory cytokines released in response to cardiac injury by chemotherapy or acute ischemia.

Synergistic down-regulation of receptor tyrosine kinases by combinations of mAbs: implications for cancer immunotherapy.

mAbs to receptor tyrosine kinases such as EGF receptor/ErbB-1 and HER2/ErbB-2 inhibit the tumorigenic growth of certain cancer cells, but although recombinant versions of such Abs are already used in oncology wards, the mechanism underlying immunotherapy remains unknown. We report that anti-EGF receptor Abs promote a slow endocytic process distinct from the rapid EGF-induced receptor internalization. Combining mAbs that engage distinct epitopes significantly accelerates receptor degradation. In addition, mAb combinations are more effective than single Abs in inhibiting HER2 signaling in vitro and tumorigenesis in animals. We present a model attributing efficacy of immunotherapy to the size of Ab-receptor lattices formed at the cell surface, which dictates the rate of endocytic clearance and extent of signaling blockade.

Epigen, the last ligand of ErbB receptors, reveals intricate relationships between affinity and mitogenicity.

Four ErbB receptors and multiple growth factors sharing an epidermal growth factor (EGF) motif underlie transmembrane signaling by the ErbB family in development and cancer. Unlike other ErbB proteins, ErbB-2 binds no known EGF-like ligand. To address the existence of a direct ligand for ErbB-2, we applied algorithms based on genomic and cDNA structures to search sequence data bases. These searches reidentified all known EGF-like growth factors including Epigen (EPG), the least characterized ligand, but failed to identify novel factors. The precursor of EPG is a widely expressed transmembrane glycoprotein that undergoes cleavage at two sites to release a soluble EGF-like domain. A recombinant EPG cannot stimulate cells singly expressing ErbB-2, but it acts as a mitogen for cells expressing ErbB-1 and co-expressing ErbB-2 in combination with the other ErbBs. Interestingly, soluble EPG is more mitogenic than EGF, although its binding affinity is 100-fold lower. Our results attribute the anomalous mitogenic power of EPG to evasion of receptor-mediated depletion of ligand molecules, as well as to inefficient receptor ubiquitylation and down-regulation. In conclusion, EPG might represent the last EGF-like growth factor and define a category of low affinity ligands, whose bioactivity differs from the more extensively studied high affinity ligands.

Prognostic significance of phosphorylated P38 mitogen-activated protein kinase and HER-2 expression in lymph node-positive breast carcinoma.

BACKGROUND: Chemotherapy-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation reportedly leads to increased apoptosis in breast carcinoma cells. The goals of the current study were to assess the incidence of activated phosphorylated p38 MAPK (P-p38) expression in invasive breast carcinoma, correlate expression of P-p38 MAPK with HER-2, and estimate the prognostic value of this marker in patients with lymph node-positive breast carcinoma treated with adjuvant chemotherapy. METHODS: P-p38, HER-2, and Ki-67 were measured using immunohistochemistry (peroxidase method) in 96 patients with lymph node-positive breast carcinoma treated with adjuvant fluorouracil, doxorubicin, and cyclophosphamide chemotherapy. All markers were measured in the primary tumors, before the initiation of adjuvant chemotherapy. The median follow-up period was 11 years after initial cancer surgery. P-p38 MAPK expression was scored visually and quantified using an image analyzer. RESULTS: The rate of P-p38 MAPK expression ranged from 19-24%, depending on the scoring system used. There was a trend toward shorter progression-free survival (PFS) for patients whose tumors expressed high levels of P-p38 MAPK, although the difference was not statistically significant (P=0.39). PFS was shorter in patients whose tumors overexpressed P-p38 MAPK and had a high level of Ki-67 (P=0.04). In HER-2-negative patients, P-p38 MAPK overexpression was associated with a shorter PFS (P=0.05). CONCLUSIONS: P38 MAPK phosphorylation occurred in 20% of primary breast carcinomas and may be associated with poor outcome in patients with lymph node-positive breast carcinoma. Further studies are needed to define the interaction between P-p38 MAPK and HER-2 expression in breast carcinoma.

Cyclin E and survival in patients with breast cancer.

BACKGROUND: Cyclin E, a regulator of the cell cycle, affects the behavior of breast-cancer cells. We investigated whether levels of cyclin E in the tumor correlated with survival among patients with breast cancer. METHODS: Tumor tissue from 395 patients with breast cancer was assayed for cyclin E, cyclin D1, cyclin D3, and the HER-2/neu oncogene with the use of Western blot analysis. Full-length, low-molecular-weight, and total cyclin E were measured. Immunohistochemical assessments of cyclin E were also made of 256 tumors. We sought correlations between levels of these molecular markers and disease-specific and overall survival. RESULTS: The median follow-up was 6.4 years. A high level of the low-molecular-weight isoforms of cyclin E, as detected by Western blotting, correlated strongly with disease-specific survival whether axillary lymph nodes were negative or positive for metastases (P<0.001). Among 114 patients with stage I breast cancer, none of the 102 patients with low levels of cyclin E in the tumor had died of breast cancer by five years after diagnosis, whereas all 12 patients with a high level of low-molecular-weight cyclin E had died of breast cancer within that period. In multivariate analysis, a high total cyclin E level or high levels of the low-molecular-weight forms of cyclin E were significantly correlated with poor outcome. The hazard ratio for death from breast cancer for patients with high total cyclin E levels as compared with those with low total cyclin E levels was 13.3--about eight times as high as the hazard ratios associated with other independent clinical and pathological risk factors. CONCLUSIONS: Levels of total cyclin E and low-molecular-weight cyclin E in tumor tissue, as measured by Western blot assay, correlate strongly with survival in patients with breast cancer.

AKT2 is frequently upregulated in HER-2/neu-positive breast cancers and may contribute to tumor aggressiveness by enhancing cell survival.

Amplification or overexpression of the HER-2/neu gene in breast cancers is associated with aggressive behavior and resistance to therapeutic regimens. The molecular mechanisms that contribute to therapeutic resistance/survival of HER-2/neu-overexpressing tumor cells have not been well defined. To determine if phosphatidylinositol 3-kinase/AKT signaling contributes to cell survival in HER-2/neu-positive breast cancers, we performed immunohistochemical analyses to evaluate expression of HER-2/neu and AKT in a series of 52 breast carcinomas. Elevated expression of HER-2/neu was found to correlate with overexpression of AKT2 protein and activation of AKT kinase. HER-2/neu-overexpressing breast cancer cell lines were resistant to apoptosis induced by UV treatment and hypoxia, which was suppressed in the presence of the phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin, indicating a link between AKT activation and stress resistance in HER-2/neu-overexpressing cells. These observations suggest that AKT signaling augments resistance to stress-induced apoptosis in breast cancer cells overexpressing HER-2/neu.