Ijeoma Adaku Umelo

Clinical activity of afatinib (BIBW 2992) in patients with lung adenocarcinoma with mutations in the kinase domain of HER2/neu

Human epidermal growth factor receptor (HER)2/neu kinase domain mutations are found in approximately 1-4% of lung adenocarcinomas with a similar phenotype to tumors with epidermal growth factor receptor (EGFR) mutations. Afatinib is a potent irreversible ErbB family blocker. We determined the tumor genomic status of the EGFR and HER2 genes in non- or light smokers with lung adenocarcinoma in patients who were entered into an exploratory Phase II study with afatinib. Five patients with a non-smoking history and metastatic lung adenocarcinomas bearing mutations in the kinase domain of HER2 gene were identified, three of which were evaluable for response. Objective response was observed in all three patients, even after failure of other EGFR- and/or HER2-targeted treatments; the case histories of these patients are described in this report. These findings suggest that afatinib is a potential novel treatment option for this subgroup of patients, even when other EGFR and HER2 targeting treatments have failed.

miR-146a Inhibits Cell Growth, Cell Migration and Induces Apoptosis in Non-Small Cell Lung Cancer Cells

Aberrant expression of microRNA-146a (miR-146a) has been reported to be involved in the development and progression of various types of cancers. However, its role in non-small cell lung cancer (NSCLC) has not been elucidated. The aim of this study was to investigate the contribution of miR-146a to various aspects of the malignant phenotype of human NSCLCs. In functional experiments, miR-146a suppressed cell growth, induced cellular apoptosis and inhibited EGFR downstream signaling in five NSCLC cell lines (H358, H1650, H1975, HCC827 and H292). miR-146a also inhibited the migratory capacity of these NSCLC cells. On the other hand, miR-146a enhanced the inhibition of cell proliferation by drugs targeting EGFR, including both TKIs (gefitinib, erlotinib, and afatinib) and a monoclonal antibody (cetuximab). These effects were independent of the EGFR mutation status (wild type, sensitizing mutation or resistance mutation), but were less potent compared to the effects of siRNA targeting of EGFR. Our results suggest that these effects of miR-146a are due to its targeting of EGFR and NF-κB signaling. We also found, in clinical formalin fixed paraffin embedded (FFPE) lung cancer samples, that low expression of miR-146a was correlated with advanced clinical TNM stages and distant metastasis in NSCLC (P<0.05). The patients with high miR-146a expression in their tumors showed longer progression-free survival (25.6 weeks in miR-146a high patients vs. 4.8 weeks in miR-146a low patients, P<0.05). miR-146a is therefore a strong candidate prognostic biomarker in NSCLC. Thus inducing miR-146a might be a therapeutic strategy for NSCLC.

Synergistic Effect of Afatinib with Su11274 in Non-Small Cell Lung Cancer Cells Resistant to Gefitinib or Erlotinib

Epidermal growth factor receptor (EGFR) and c-MET receptors are expressed on many non-small cell lung cancer (NSCLC) cells. Current single agent therapeutic targeting of a mutant EGFR has a high efficacy in the clinic, but is not curative. Here, we investigated the combination of targeting EGFR and c-MET pathways in NSCLC cells resistant to receptor tyrosine kinase inhibitors (TKIs), using RNA interference and inhibition by TKIs. Different NSCLC cell lines with various genomic characteristics (H358, H1650 and H1975) were transfected with EGFR-specific-siRNA, T790M-specific-siRNA, c-MET siRNA or the combination. Subsequently EGFR TKIs (gefitinib, erlotinib or afatinib) or monoclonal antibody cetuximab were combined respectively with the c-MET-specific TKI su11274 in NSCLC cell lines. The cell proliferation, viability, caspase−3/7 activity and apoptotic morphology were monitored by spectrophotometry, fluorimetry and fluorescence microscopy. The combined effect of EGFR TKIs, or cetuximab and su11274, was evaluated using a combination index. The results showed that the cell lines that were relatively resistant to EGFR TKIs, especially the H1975 cell line containing the resistance T790M mutation, were found to be more sensitive to EGFR-specific-siRNA. The combination of EGFR siRNA plus c-MET siRNA enhanced cell growth inhibition, apoptosis induction and inhibition of downstream signaling in EGFR TKI resistant H358, H1650 and H1975 cells, despite the absence of activity of the c-MET siRNA alone. EGFR TKIs or cetuximab plus su11274 were also consistently superior to either agent alone. The strongest biological effect was observed when afatinib, an irreversible pan-HER blocker was combined with su11274, which achieved a synergistic effect in the T790M mutant H1975 cells. In a conclusion, our findings offer preclinical proof of principle for combined inhibition as a promising treatment strategy for NSCLC, especially for patients in whom current EGFR-targeted treatments fail due to the presence of the T790M-EGFR-mutation or high c-MET expression.

Effect of siRNAs targeting the EGFR T790M mutation in a non-small cell lung cancer cell line resistant to EGFR tyrosine kinase inhibitors and combination with various agents.

The epidermal growth factor receptor (EGFR) is a validated therapeutic target in non-small cell lung cancer (NSCLC). However, some mutations confer resistance to current available agents, especially the frequently occurring T790M mutation. In the current study, we have examined, in a NSCLC cell line H1975 containing both L858R and T790M mutations, the effect of T790M-specific-siRNAs versus other EGFR-specific-siRNAs. T790M-specific-siRNAs were able to inhibit T790M and EGFR mRNA, to reduce EGFR protein expression, as well as to reduce the cell growth and induce cell caspase activity in H1975 cells. However, this effect showed less potency compared to the other EGFR-specific-siRNAs. EGFR-specific-siRNAs strongly inhibited cell growth and induced apoptosis in H358, H1650, H292, HCC827 and also in H1975 cells, which showed weak response to tyrosine kinase inhibitors (TKIs) or cetuximab. The addition of T790M-specific-siRNAs could rescue the sensitivity of T790M mutant H1975 cells to TKIs. The combination of T790M-specific-siRNAs and cetuximab also additively enhanced cell growth inhibition and induction of apoptosis in H1975 cells. Among the anti-EGFR agents tested, the strongest biological effect was observed when afatinib was combined with T790M-specific-siRNAs. Afatinib also offered extra effect when combined with cetuximab in H1975 cells. In conclusion, knock-down of T790M transcript by siRNAs further decreases the cell growth of T790M mutant lung cancer cells that are treated with TKIs or cetuximab. The combination of a potent, irreversible kinase inhibitor such as afatinib, with T790M-specific-siRNAs should be further investigated as a new strategy in the treatment of lung cancer containing the resistant T790M mutation.

Quantification of epidermal growth factor receptor T790M mutant transcripts in lung cancer cells by real-time reverse transcriptase–quantitative polymerase chain reaction

A simple and sensitive real-time reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was developed to quantify threonine-to-methionine substitution at amino acid position 790 (T790M) mutant transcripts in a wild-type (wt) epidermal growth factor receptor background. The assay is based on three unmodified oligonucleotides, and both SYBR Green and a Taqman probe can be used. To increase the discrimination between mutant and wt signals, ARMS (amplification refractory mutation system) and LNA (locked nucleic acid) primers were tested, but a benefit was observed only with plasmids and not with cellular complementary DNA. The RT-qPCR assay using transcript-specific primers can detect as few as 1% T790M transcripts in a wt background and, therefore, will be useful in RNA interference studies specifically targeting mutant RNA.

Stromal Modulators of TGF-β in Cancer

Transforming growth factor-β (TGF-β) is an intriguing cytokine exhibiting dual activities in malignant disease. It is an important mediator of cancer invasion, metastasis and angiogenesis, on the one hand, while it exhibits anti-tumor functions on the other hand. Elucidating the precise role of TGF-β in malignant development and progression requires a better understanding of the molecular mechanisms involved in its tumor suppressor to tumor promoter switch. One important aspect of TGF-β function is its interaction with proteins within the tumor microenvironment. Several stromal proteins have the natural ability to interact and modulate TGF-β function. Understanding the complex interplay between the TGF-β signaling network and these stromal proteins may provide greater insight into the development of novel therapeutic strategies that target the TGF-β axis. The present review highlights our present understanding of how stroma modulates TGF-β activity in human cancers.

Identification of a novel HER3 activating mutation homologous to EGFR-L858R in lung cancer.

Somatic mutations found within the tyrosine kinase domain (TKD) of the human epidermal growth factor (HER) family of receptors have been implicated in the development and progression of non-small cell lung cancer (NSCLC). However, no conclusive reports have described pathogenic mutations in kinase-impaired HER3. Here, we report a case of an advanced chemotherapy-resistant NSCLC, harboring a novel HER3V855A somatic mutation homologous to the EGFRL858Ractivating mutation. Co-expression of HER3V855A and wild-type HER2 enhances ligand-induced transformation of murine and human cell lines, while HER-targeted inhibitors potently suppress mutant HER3 activity. Consistent with these observations, in silico computational modeling predicts that mutant V855A alters the kinase domain and c-terminal end of the HER3 protein. Taken together, these findings provide a basis for the clinical exploration of targeted therapies in HER3 mutant NSCLC and by extrapolation, in other cancers that more frequently carry somatic HER3 mutations.

Combined inhibition of rho-associated protein kinase and EGFR suppresses the invasive phenotype in EGFR-dependent lung cancer cells

INTRODUCTION Lung cancer remains the leading cause of cancer-related mortality worldwide, with metastatic disease frequently a prominent feature at the time of diagnosis. The role of NSCLC-derived EGFR mutations in cancer cell proliferation and survival has been widely reported, but little is known about the function of these mutations in invasive growth and metastasis. In this study, we sought to evaluate the intrinsic invasive properties of NSCLC cells with differing EGFR status and examine possible therapeutic targets that can abrogate invasive growth. MATERIALS AND METHODS Collagen-based assays and 3D cell cultures were used to assess morphological features, actin cytoskeleton dynamics and the invasive capacity of NSCLC cell lines with differing EGFR status. The role of the RhoA/ROCK/MYPT1 and EGFR/HER pathways in NSCLC-related invasion was investigated by pharmacological inhibition and RNA interference techniques. RESULTS We demonstrate a positive correlation between EGFR mutational/amplification status and invasive capacity. Knockdown of wild-type and mutant EGFR leads to depletion of active and total MYPT1 levels. Combined pharmacological inhibition or genetic ablation of ROCK/EGFR suppresses the hallmarks of cancer cells and abrogates the invasive phenotype in EGFR-dependent NSCLC cells. CONCLUSIONS These observations suggest that combined targeting of the ROCK and EGFR/HER pathways may be a potential therapeutic approach in limiting invasive growth in NSCLC.

Targeting Polo-like kinase 1 and TRAIL enhances apoptosis in non-small cell lung cancer

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in cancer cells without causing damage to normal cells. However, some tumors are resistant to TRAIL monotherapy and clinical studies assessing targeted agents towards the TRAIL receptor have failed to show robust therapeutic activity. Evidence has shown that standard anti-mitotic drugs can induce synergistic apoptosis upon combination with TRAIL via cell cycle arrest. Polo like kinase-1 (PLK1) plays a critical role in different stages of cell cycle progression and mitosis. A number of investigations have demonstrated that PLK1 inhibition causes cell cycle arrest and mitotic catastrophe in non-small cell lung cancer (NSCLC), and we thus postulated that PLK1 inhibition could enhance TRAIL-induced apoptosis. We demonstrate that the combination of a TRAIL receptor agonist and a PLK1 inhibitor synergistically reduces cell viability, and strongly increases apoptosis in NSCLC cellular models. Consistent with our in vitro observations, this drug combination also significantly reduces tumor growth in vivo. Our data additionally reveal that G2/M cell cycle arrest and downregulation of Mcl-1 and signal transducer and activator of transcription 3 (STAT3) activity following PLK1 inhibition may contribute to the sensitization of TRAIL-induced apoptosis in NSCLC. Together, these data support the further exploration of combined TRAIL and PLK1 inhibition in the treatment of NSCLC.

Innovative methods for biomarker discovery in the evaluation and development of cancer precision therapies

The discovery of biomarkers able to detect cancer at an early stage, to evaluate its aggressiveness, and to predict the response to therapy remains a major challenge in clinical oncology and precision medicine. In this review, we summarize recent achievements in the discovery and development of cancer biomarkers. We also highlight emerging innovative methods in biomarker discovery and provide insights into the challenges faced in their evaluation and validation.