Different morphology, stage and treatment affect immune cell infiltration and long-term outcome in patients with non-small-cell lung carcinoma

Aims: Development of effective immune-based therapies for patients with non-small-cell lung carcinoma (NSCLC) depends on an accurate characterization of complex interactions that occur between immune cells and the tumour environment. Methods and results: Innate and adaptive immune responses were evaluated in relation to prognosis in 65 patients with surgically excised NSCLC. Immunohistochemistry and morphometry were used to determine the abundance and distribution of immune cells. We found low numbers of immune cells and levels of cytokines in the tumour environment when compared with surrounding parenchyma. Smoking was associated inversely with the adaptive immune response and directly with innate immunity. We observed a prominent adaptive immune response in squamous cell carcinomas (SCC) but greater innate immune responses in adenocarcinomas and large cell carcinomas. Cox model analysis showed a low risk of death for smoking <41 packs/year, N-0 tambour stage, squamous carcinoma, CD4(+) > 16.81% and macrophages/monocytes >4.5%. Collectively, the data indicate that in NSCLC there is not a substantive local immune cell infiltrate within the tumour. Conclusion: Although immune cell infiltration is limited in NSCLC it appears to have an impact on prognosis and this may be of relevance for new immunotherapeutic approaches.

Central Granular Cell Odontogenic Tumor of the Maxilla

The central granular cell odontogenic tumor (CGCOT) is a rare benign odontogenic neoplasm composed of varying amounts of large eosinophilic granular cells and apparently inactive odontogenic epithelium. It tends to occur as a small asymptomatic swelling in the posterior region of the mandible with nonaggressive appearance. We report an unusual case of CGCOT in the maxillary region with clinical features of malignancy. The patient underwent surgical treatment, and the 2-year follow-up revealed no signs of recurrence. Central granular cell odontogenic tumor is a very rare condition with few cases reported, especially in the maxillary region. This case highlights the possibility of aggressive behavior by these lesions.

Independent of ErbB1 gene copy number, EGF stimulates migration but is not associated with cell proliferation in non-small cell lung cancer

Abstract Background Lung cancer often exhibits molecular changes, such as the overexpression of the ErbB1 gene. ErbB1 encodes epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, involved mainly in cell proliferation and survival. EGFR overexpression has been associated with more aggressive disease, poor prognosis, low survival rate and low response to therapy. ErbB1 amplification and mutation are associated with tumor development and are implicated in ineffective treatment. The aim of the present study was to investigate whether the ErbB1 copy number affects EGFR expression, cell proliferation or cell migration by comparing two different cell lines. Methods The copies of ErbB1 gene was evaluated by FISH. Immunofluorescence and Western blotting were performed to determine location and expression of proteins mentioned in the present study. Proliferation was studied by flow cytometry and cell migration by wound healing assay and time lapse. Results We investigated the activation and function of EGFR in the A549 and HK2 lung cancer cell lines, which contain 3 and 6 copies of ErbB1, respectively. The expression of EGFR was lower in the HK2 cell line. EGFR was activated after stimulation with EGF in both cell lines, but this activation did not promote differences in cellular proliferation when compared to control cells. Inhibiting EGFR with AG1478 did not modify cellular proliferation, confirming previous data. However, we observed morphological alterations, changes in microfilament organization and increased cell migration upon EGF stimulation. However, these effects did not seem to be consequence of an epithelial-mesenchymal transition. Conclusion EGFR expression did not appear to be associated to the ErbB1 gene copy number, and neither of these aspects appeared to affect cell proliferation. However, EGFR activation by EGF resulted in cell migration stimulation in both cell lines.

High-throughput sequencing of small RNA transcriptomes reveals critical biological features targeted by microRNAs in cell models used for squamous cell cancer research

Abstract Background The implication of post-transcriptional regulation by microRNAs in molecular mechanisms underlying cancer disease is well documented. However, their interference at the cellular level is not fully explored. Functional in vitro studies are fundamental for the comprehension of their role; nevertheless results are highly dependable on the adopted cellular model. Next generation small RNA transcriptomic sequencing data of a tumor cell line and keratinocytes derived from primary culture was generated in order to characterize the microRNA content of these systems, thus helping in their understanding. Both constitute cell models for functional studies of microRNAs in head and neck squamous cell carcinoma (HNSCC), a smoking-related cancer. Known microRNAs were quantified and analyzed in the context of gene regulation. New microRNAs were investigated using similarity and structural search, ab initio classification, and prediction of the location of mature microRNAs within would-be precursor sequences. Results were compared with small RNA transcriptomic sequences from HNSCC samples in order to access the applicability of these cell models for cancer phenotype comprehension and for novel molecule discovery. Results Ten miRNAs represented over 70% of the mature molecules present in each of the cell types. The most expressed molecules were miR-21, miR-24 and miR-205, Accordingly; miR-21 and miR-205 have been previously shown to play a role in epithelial cell biology. Although miR-21 has been implicated in cancer development, and evaluated as a biomarker in HNSCC progression, no significant expression differences were seen between cell types. We demonstrate that differentially expressed mature miRNAs target cell differentiation and apoptosis related biological processes, indicating that they might represent, with acceptable accuracy, the genetic context from which they derive. Most miRNAs identified in the cancer cell line and in keratinocytes were present in tumor samples and cancer-free samples, respectively, with miR-21, miR-24 and miR-205 still among the most prevalent molecules at all instances. Thirteen miRNA-like structures, containing reads identified by the deep sequencing, were predicted from putative miRNA precursor sequences. Strong evidences suggest that one of them could be a new miRNA. This molecule was mostly expressed in the tumor cell line and HNSCC samples indicating a possible biological function in cancer. Conclusions Critical biological features of cells must be fully understood before they can be chosen as models for functional studies. Expression levels of miRNAs relate to cell type and tissue context. This study provides insights on miRNA content of two cell models used for cancer research. Pathways commonly deregulated in HNSCC might be targeted by most expressed and also by differentially expressed miRNAs. Results indicate that the use of cell models for cancer research demands careful assessment of underlying molecular characteristics for proper data interpretation. Additionally, one new miRNA-like molecule with a potential role in cancer was identified in the cell lines and clinical samples.

Small Molecules as Modulators of Different Targets Involved in Tumor Progression

Tumor is a lesion that may be formed by an abnormal growth of neoplastic cells. Many factors increase the risk of cancer and different targets are involved in tumor progression. Within this thesis, we have addressed two different biological targets, independently connected with tumor formation, e.g. Hsp90 and androgen receptor. The ATP-dependent chaperone Hsp90 is responsible for the conformational maturation and the renaturation of proteins. “Client” proteins are associated with the cancer hallmarks, as cell proliferation and tumor progression. Consequently, Hsp90 has evolved into promising anticancer target. Over the past decade, radicicol has been identified as potential anticancer agent targeting Hsp90, but it is not active in vivo. With that aim of obtaining radicicol-related derivatives, we developed the design and synthesis of new chalcones analogs. Chalcones, which are abundant in edible plants, own a diverse array of pharmacological activities and are considered a versatile scaffold for drug design. Antiproliferative assays and western blot analysis on the new compounds showed that some of those display an interesting cytotoxic effect and the ability to modulate Hsp90 client proteins expression. Androgen Receptor (AR) hypersensitivity plays crucial role in prostate cancer, which progression is stimulated by androgens. The therapy consists in a combination of surgical or chemical castration, along with antiandrogens treatment. Casodex® (bicalutamide), is the most widespread antiandrogen used in clinic. However, hormonal therapy is time-limited since many patients develop resistance. Commercially available antiandrogens show a common scaffold, e.g. two substituted aromatic rings linked by a linear or a cyclic spacer. With the aim of obtaining novel pure AR antagonists, we developed a new synthetic methodology, which allowed us to introduce, as linker between two suitably chosen aromatic rings, a triazole moiety. Preliminary data suggest that the herein reported new molecules generally decrease PSA expression, thus confirming their potential AR antagonistic activity.

Generation and characterization of mouse models of Small cell lung cancer and Basal cell carcinoma for the preclinical evaluation of new therapies

Background. Human small cell lung cancer (SCLC) accounting for approximately 15-20% of all lung cancers, is an aggressive tumor with high propensity for early regional and distant metastases. Although the initial tumor rate response to chemotherapy is very high, SCLC relapses after approximately 4 months in ED and 12 months in LD. Basal cell carcinoma (BCC) is the most prevalent cancer in the western world, and its incidence is increasing worldwide. This type of cancer rarely metastasizes and the death rate is extraordinary low. Surgery is curative for most of the patients, but for those that develop locally advanced or metastatic BCC there is currently no effective treatment. Both types of cancer have been deeply investigated and genetic alterations, MYCN amplification (MA) among the most interesting, have been found. These could become targets of new pharmacological therapies. Procedures. We created and characterized novel BLI xenograft orthotopic mouse models of SCLC to evaluate the tumor onset and progression and the efficacy of new pharmacological strategies. We compared an in vitro model with a transgenic mouse model of BCC, to investigate and delineate the canonical HH signalling pathway and its connections with other molecular pathways. Results and conclusions. The orthotopic models showed latency and progression patterns similar to human disease. Chemotherapy treatments improved survival rates and validated the in vivo model. The presence of MA and overexpression were confirmed in each model and we tested the efficacy of a new MYCN inhibitor in vitro. Preliminar data of BCC models highlighted Hedgehog pathway role and underlined the importance of both in vitro and in vivo strategies to achieve a better understanding of the pathology and to evaluate the applicability of new therapeutic compounds

Mechanistic study of cell death induction by O 6-methylating agents, focusing on the role of homologous recombination as a molecular target for sensitization of tumor cells to chemotherapeutic alkylating agents

Chemotherapeutic SN1‑methylating agents are important anticancer drugs. They induce several covalent modifications in the DNA, from which O6‑methylguanine (O6MeG) is the main toxic lesion. In this work, different hypotheses that have been proposed to explain the mechanism of O6MeG‑triggered cell death were tested. The results of this work support the abortive processing model, which states that abortive post‑replicative processing of O6MeG‑driven mispairs by the DNA mismatch repair (MMR) machinery results in single‑strand gaps in the DNA that, upon a 2nd round of DNA replication, leads to DNA double‑strand break (DSB) formation, checkpoint activation and cell death. In this work, it was shown that O6MeG induces an accumulation of cells in the 2nd G2/M‑phase after treatment. This was accompanied by an increase in DSB formation in the 2nd S/G2/M‑phase, and paralleled by activation of the checkpoint kinases ATR and CHK1. Apoptosis was activated in the 2nd cell cycle. A portion of cells continue proliferating past the 2nd cell cycle, and triggers apoptosis in the subsequent generations. An extension to the original model is proposed, where the persistence of O6MeG in the DNA causes new abortive MMR processing in the 2nd and subsequent generations, where new DSB are produced triggering cell death. Interestingly, removal of O6MeG beyond the 2nd generation lead to a significant, but not complete, reduction in apoptosis, pointing to the involvement of additional mechanisms as a cause of apoptosis. We therefore propose that an increase in genomic instability resulting from accumulation of mis‑repaired DNA damage plays a role in cell death induction. Given the central role of DSB formation in toxicity triggered by chemotherapeutic SN1‑alkylating agents, it was aimed in the second part of this thesis to determine whether inhibition of DSB repair by homologous recombination (HR) or non‑homologous end joining (NHEJ) is a reasonable strategy for sensitizing glioblastoma cells to these agents. The results of this work show that HR down‑regulation in glioblastoma cells impairs the repair of temozolomide (TMZ)‑induced DSB. HR down‑regulation greatly sensitizes cells to cell death following O6‑methylating (TMZ) or O6‑chlorethylating (nimustine) treatment, but not following ionizing radiation. The RNAi mediated inhibition in DSB repair and chemo‑sensitization was proportional to the knockdown of the HR protein RAD51. Chemo‑sensitization was demonstrated for several HR proteins, in glioma cell lines proficient and mutated in p53. Evidence is provided showing that O6MeG is the primary lesion responsible for the increased sensitivity of glioblastoma cells following TMZ treatment, and that inhibition of the resistance marker MGMT restores the chemo‑sensitization achieved by HR down‑regulation. Data are also provided to show that inhibition of DNA‑PK dependent NHEJ does not significantly sensitized glioblastoma cells to TMZ treatment. Finally, the data also show that PARP inhibition with olaparib additionally sensitized HR down‑regulated glioma cells to TMZ. Collectively, the data show that processing of O6MeG through two rounds of DNA replication is required for DSB formation, checkpoint activation and apoptosis induction, and that O6MeG‑triggered apoptosis is also executed in subsequent generations. Furthermore, the data provide proof of principle evidence that down‑regulation of HR is a reasonable strategy for sensitizing glioma cells to killing by O6‑alkylating chemotherapeutics.

The stem cell gene "inhibitor of differentiation 1" (ID1) is frequently expressed in non-small cell lung cancer

Inhibitor of differentiation 1 (ID1) plays a role in cellular differentiation, proliferation, angiogenesis and tumor invasion. As shown recently, ID1 is positively regulated by the tyrosine kinase SRC in lung carcinoma cell lines and with that appears as a potential new therapeutic target in non-small cell carcinoma (NSCLC). To substantiate this hypothesis we examined ID1, SRC and matrix metalloproteinase-9 (MMP-9) immunohistochemically in human NSCLC specimens.

Cyclin D1 (CCND1) A870G gene polymorphism modulates smoking-induced lung cancer risk and response to platinum-based chemotherapy in non-small cell lung cancer (NSCLC) patients

PURPOSE: The cyclin D1 (CCND1) A870G gene polymorphism is linked to the outcome in patients with resectable non-small cell lung cancer (NSCLC). Here, we investigated the impact of this polymorphism on smoking-induced cancer risk and clinical outcome in patients with NSCLC stages I-IV. METHODS: CCND1 A870G genotype was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis (RFLP) of DNA extracted from blood. The study included 244 NSCLC patients and 187 healthy control subjects. RESULTS: Patient characteristics were: 70% male, 77% smokers, 43% adenocarcinoma, and 27% squamous cell carcinoma. Eighty-one percent of the patients had stages III-IV disease. Median age at diagnosis was 60 years and median survival was 13 months. Genotype frequencies of patients and controls both conformed to the Hardy Weinberg equilibrium. The GG genotype significantly correlated with a history of heavy smoking (>or=40 py, P=0.02), and patients with this genotype had a significantly higher cigarette consumption than patients with AA/AG genotypes (P=0.007). The GG genotype also significantly correlated with tumor response or stabilization after a platinum-based first-line chemotherapy (P=0.04). Survival analysis revealed no significant differences among the genotypes. CONCLUSION: Evidence was obtained that the CCND1 A870G gene polymorphism modulates smoking-induced lung cancer risk. Further studies are required to explore the underlying molecular mechanisms and to test the value of this gene polymorphism as a predictor for platinum-sensitivity in NSCLC patients.

Cardiac glycosides initiate Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cells by up-regulation of death receptors 4 and 5

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the TNF family known to transduce their death signals via cell membrane receptors. Because it has been shown that Apo2L/TRAIL induces apoptosis in tumor cells without or little toxicity to normal cells, this cytokine became of special interest for cancer research. Unfortunately, cancer cells are often resistant to Apo2L/TRAIL-induced apoptosis; however, this can be at least partially negotiated by parallel treatment with other substances, such as chemotherapeutic agents. Here, we report that cardiac glycosides, which have been used for the treatment of cardiac failure for many years, sensitize lung cancer cells but not normal human peripheral blood mononuclear cells to Apo2L/TRAIL-induced apoptosis. Sensitization to Apo2L/TRAIL mediated by cardiac glycosides was accompanied by up-regulation of death receptors 4 (DR4) and 5 (DR5) on both RNA and protein levels. The use of small interfering RNA revealed that up-regulation of death receptors is essential for the demonstrated augmentation of apoptosis. Blocking of up-regulation of DR4 and DR5 alone significantly reduced cell death after combined treatment with cardiac glycosides and Apo2L/TRAIL. Combined silencing of DR4 and DR5 abrogated the ability of cardiac glycosides and Apo2L/TRAIL to induce apoptosis in an additive manner. To our knowledge, this is the first demonstration that glycosides up-regulate DR4 and DR5, thereby reverting the resistance of lung cancer cells to Apo2/TRAIL-induced apoptosis. Our data suggest that the combination of Apo2L/TRAIL and cardiac glycosides may be a new interesting anticancer treatment strategy.

Early clinical trial experience with vaccine therapies in non-small-cell lung cancer

Cancer immunotherapy has made great progress because of advances in immunology and molecular biology. Increased understanding of mechanisms by which lung cancer cells escape the immune system and recognition of key tumor antigens and immune system components involved in tumor ignorance have led to the development of a variety of lung cancer vaccines. Immunotherapy has advanced from using nonspecific immunomodulatory agents to lung cancer-specific tumor antigens and tumor cell-derived vaccines. While understanding of immune processes and malignancy has improved, there is great opportunity for further research of vaccine therapies in non-small-cell lung cancer. Herein, we review the development and evolution of early lung cancer vaccine trials.

OCT4 expression in human non-small cell lung cancer: implications for therapeutic intervention

Here we investigate the expression of OCT4 human lung adenocarcinoma and bronchioloalveolar carcinoma (BAC) tumor biopsies and tumor-derived primary cell cultures. OCT4 has been detected in several human tumors suggesting a potentially critical role in tumorigenesis. We assessed the presence of OCT4 in clinical tumor samples of both adenocarcinoma and BAC at the cellular and transcriptional levels, respectively. Furthermore, we evaluated tumor-derived cell cultures for potential differences in OCT4 expression. Immunohistochemical analysis depicted OCT4 in 2 of 8 adenocarcinoma tumor samples and 3 of 5 BAC tumor samples, with no apparent difference in the degree of expression among the sections examined. These results were validated by transcript analysis. Flow cytometric assessment of 11 adenocarcinoma-derived cell cultures and 3 BAC-derived cell cultures revealed significantly higher OCT4 expression in adenocarcinoma tumors compared to their normal counterparts. This, however, was not observed in the BAC cultures. Comparative studies of OCT4 in adenocarcinoma and BAC tumor cell cultures demonstrated a dramatically higher expression in the former. The expression of OCT4 may represent a specific and effective target for therapeutic intervention in adenocarcinoma and BAC. In addition, the aberrant expression and distribution of OCT4 may indicate important parameters concerning the differences between adenocarcinoma and BAC.

AKT/mTOR pathway activation and BCL-2 family proteins modulate the sensitivity of human small cell lung cancer cells to RAD001

PURPOSE: The Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancers and plays an important role in small cell lung cancer (SCLC) biology. We investigated the potential of targeting mTOR signaling as a novel antitumor approach in SCLC. EXPERIMENTAL DESIGN: The expression of mTOR in patient specimens and in a panel of SCLC cell lines was analyzed. The effects on SCLC cell survival and downstream signaling were determined following mTOR inhibition by the rapamycin derivative RAD001 (Everolimus) or down-regulation by small interfering RNA. RESULTS: We found elevated expression of mTOR in patient specimens and SCLC cell lines, compared with normal lung tissue and normal lung epithelial cells. RAD001 treatment impaired basal and growth factor-stimulated cell growth in a panel of SCLC cell lines. Cells with increased Akt pathway activation were more sensitive to RAD001. Accordingly, a constitutive activation of the Akt/mTOR pathway was sufficient to sensitize resistant SCLC cells to the cytotoxic effect of RAD001. In the sensitive cells, RAD001 showed a strong additive effect to the proapoptotic action of the chemotherapeutic agent etoposide. Intriguingly, we observed low Bcl-2 family proteins levels in the SCLC cells with a constitutive Akt pathway activation, whereas an increased expression was detected in the RAD001-resistant SCLC cells. An antisense construct targeting Bcl-2 or a Bcl-2-specific inhibitor was able to sensitize resistant SCLC cells to RAD001. Moreover, SCLC tumor growth in vivo was significantly inhibited by RAD001. CONCLUSION: Together, our data show that inhibiting mTOR signaling with RAD001 potently disrupts growth and survival signaling in human SCLC cells.

Preserving middle lobe to improve lung function in non-small-cell lung cancer

When a lung tumor arises in segment 6, the close anatomical relationship to the middle lobe bronchus may make a lower bilobectomy necessary. Sleeve lobectomy may be an alternative. These procedures were compared retrospectively in 36 patients operated on between January 2005 and December 2006 with non-small-cell lung cancer (stage I-IIIB) of the right lower lobe. Sleeve lobectomy was performed in 21 patients and bilobectomy in 15 (41%). Preoperative lung function was comparable in both groups. Radical resection was achieved in 34/36 patients. Operation time was 121 min for sleeve lobectomy and 144 min for bilobectomy. Chest tubes were removed after 5 days in both groups. Postoperative lung function was better after sleeve lobectomy than bilobectomy (forced expiratory volume in 1st sec: 78% vs. 69%). Preservation of the middle lobe by sleeve lobectomy is feasible. There was no evidence that this resection was less radical, and complication rates were similar in both groups.

EGFR exon-level biomarkers of the response to bevacizumab/erlotinib in non-small cell lung cancer

Activating epidermal growth factor receptor (EGFR) mutations are recognized biomarkers for patients with metastatic non-small cell lung cancer (NSCLC) treated with EGFR tyrosine kinase inhibitors (TKIs). EGFR TKIs can also have activity against NSCLC without EGFR mutations, requiring the identification of additional relevant biomarkers. Previous studies on tumor EGFR protein levels and EGFR gene copy number revealed inconsistent results. The aim of the study was to identify novel biomarkers of the response to TKIs in NSCLC by investigating whole genome expression at the exon-level. We used exon arrays and clinical samples from a previous trial (SAKK19/05) to investigate the expression variations at the exon-level of 3 genes potentially playing a key role in modulating treatment response: EGFR, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and vascular endothelial growth factor (VEGFA). We identified the expression of EGFR exon 18 as a new predictive marker for patients with untreated metastatic NSCLC treated with bevacizumab and erlotinib in the first line setting. The overexpression of EGFR exon 18 in tumor was significantly associated with tumor shrinkage, independently of EGFR mutation status. A similar significant association could be found in blood samples. In conclusion, exonic EGFR expression particularly in exon 18 was found to be a relevant predictive biomarker for response to bevacizumab and erlotinib. Based on these results, we propose a new model of EGFR testing in tumor and blood.