Non-small Cell Lung Cancer Tumor Growth Research Articles

The emerging role of T cell cytokines in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is among the most frequent types of cancer and associated with significant mortality. Recent studies suggest that tumor infiltrating T lymphocytes and their immunoregulatory cytokines play a fundamental role in controlling tumor growth and metastasis. Whereas T helper 1 cells and their master transcription factor T-bet have been identified as important regulators of IFN-gamma driven anti-tumoral immune response, IL-17 producing T helper 17 cells expressing RORA/C transcription factors have been found to augment tumor growth and cell proliferation in NSCLC. Furthermore, regulatory T cells were shown to impair immune responses against lung cancer. As modulation of T cell cytokines such as IL-17A has been recently shown to suppress tumor growth in experimental models, anti-cytokine agents emerge as a promising new approach for treatment of lung cancer.

Non-small cell lung cancer: the era of targeted therapy.

In this review, the authors aim to provide an overview of current molecular targeted therapies for NSCLC, to propose an algorithm for clinical application of presently available treatment strategies, and to identify future directions for this important area of research. Historically, choice of treatment algorithm for the management of non-small cell lung cancer (NSCLC) has relied heavily upon histology and clinical staging information, typically assigning patients to surgery, chemotherapy, radiation, or a combination thereof. However, previous treatment strategies have been fraught with disappointing response rates and significant systemic toxicities. The concept of personalized therapy for NSCLC involves characterization of each individual patient's tumor, in terms of genetic aberrations and expected biologic behavior, and using this information to tailor subsequent clinical management. Several driver mutations have been identified to date in subsets of patients with NSCLC, and, by focusing on specific molecular targets, new agents have been developed with the intent of treating the cancer cells while causing minimal toxicity to benign, healthy cells. In particular, current strategies exist to identify patients with epidermal growth factor receptor gene mutations and anaplastic lymphoma kinase rearrangements, with promising results upon clinical application of agents targeting these abnormalities. Moving forward, attempts are being made to determine comprehensive genetic and biologic characterization of individuals' NSCLC tumors and to incorporate these findings into everyday practice. The era of targeted therapy is upon us. As we seek to expand our knowledge of the specific molecular and cellular derangements leading to growth and proliferation of NSCLC tumors, our efforts bring us closer to ultimately providing each patient with a personalized plan of care.

Radiation/Paclitaxel Treatment of p53-Abnormal Non-Small Cell Lung Cancer Xenograft Tumor and Associated Mechanism

Mutations in key tumor suppressor genes such as tumor protein 53 (TP53) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) are the main genetic alterations in cancers. TP53 mutations have been found in most patients with non-small cell lung cancer (NSCLC), whereas PTEN mutations are rarely found in lung cancer, though most NSCLCs lack PTEN protein synthesis. However, the signaling involved in radio- and chemotherapy of NSCLC with wild-type PTEN and nonfunctional p53 is not clearly understood. In this study, we established a xenograft tumor model with H358 NSCLC cells expressing wild-type PTEN, but nonfunctional p53. Protein expression and phosphorylation of PTEN and its downstream signal molecules in NSCLC tissues were detected by Western blot. We demonstrated that radiation and paclitaxel alone inhibited tumor growth, but a combined therapy of radiation and paclitaxel was more effective in inhibiting NSCLC tumor growth. Interestingly, both radiation and paclitaxel significantly increased PTEN protein expression and phosphorylation. Further identification of the affected PTEN downstream molecules showed that Akt phosphorylation at Ser(473) and Thr(308) residues was significantly decreased, whereas Bax and cleaved caspase-3 levels were significantly increased in tumor tissues treated with both radiation and paclitaxel. The combined treatment was more effective than either treatment alone in regulating the studied molecules. We also found that paclitaxel, but not radiation, inhibited phosphoinositide 3-kinase (PI3K) activity. Our study suggested that a PTEN-PI3K-Akt-Bax signaling cascade is involved in the therapeutic effect of combined radiation/paclitaxel treatment in NSCLC without p53 expression. Our study also suggested that PTEN is an ideal target in tumors with wild-type PTEN and a lack of functional p53.

L1 Cell Adhesion Molecule Promotes Tumorigenicity and Metastatic Potential in Non–Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) is a highly metastatic cancer with limited treatment options, thus requiring development of novel targeted therapies. Our group previously identified L1 cell adhesion molecule (L1CAM) expression as a member of a prognostic multigene expression signature for NSCLC patients. However, there is little information on the biologic function of L1CAM in lung cancer cells. This study investigates the functional and prognostic role of L1CAM in NSCLC. Cox proportional hazards regression analysis was done on four independent published mRNA expression datasets of primary NSCLCs. L1CAM expression was suppressed by short-hairpin RNA (shRNA)-mediated silencing in human NSCLC cell lines. Effects were assessed by examining in vitro migration and invasion, in vivo tumorigenicity in mice, and metastatic potential using an orthotopic xenograft rat model of lung cancer. L1CAM is an independent prognostic marker in resected NSCLC patients, with overexpression strongly associated with worse prognosis. L1CAM downregulation significantly decreased cell motility and invasiveness in lung cancer cells and reduced tumor formation and growth in mice. Cells with L1CAM downregulation were deficient in constitutive extracellular signal-regulated kinase (Erk) activation. Orthotopic studies showed that L1CAM suppression in highly metastatic lung cancer cells significantly decreases spread to distant organs, including bone and kidney. L1CAM is a novel prometastatic gene in NSCLC, and its downregulation may effectively suppress NSCLC tumor growth and metastasis. Targeted inhibition of L1CAM may be a novel therapy for NSCLC.

RNAi-mediated A disintegrin and metalloproteinase 9 gene silencing inhibits the tumor growth of non-small lung cancer in vitro and in vivo.

A disintegrin and metalloproteinase 9 (ADAM9) is a type I transmembrane protein that has been associated with cancer development and metastasis in various types of cancer. However, little is known about its role in non-small cell lung cancer (NSCLC). The aim of the present study was to evaluate whether downregulation of ADAM9 affects cell proliferation, apoptosis, migration and invasion in NSCLC. Thus, a recombinant lentiviral small hairpin RNA expression vector carrying ADAM9 was constructed and infected into the human NSCLC cell line A549. Cell proliferation, apoptosis, migration and invasion in vitro and tumor growth in vivo were determined following downregulation of ADAM9 by RNA interference-mediated ADAM9 gene silencing. It was found that downregulation of ADAM9 expression using an RNA silencing approach in A549 tumor cells significantly inhibited cell proliferation, migration and invasion, induced cell apoptosis in vitro, as well as suppressed in vivo tumor growth in an experimental mouse model of lung metastasis. These data indicate that ADAM9 is potentially an important new therapeutic target for the prevention of tumor growth in NSCLC.

Polysaccharide of Cordyceps sinensis Enhances Cisplatin Cytotoxicity in Non–Small Cell Lung Cancer H157 Cell Line

The extracts of Cordyceps sinensis (Berk) Sacc (CS) have been used as a traditional medicine for centuries. However, few studies have examined the adjuvant action of CS in the treatment of non-small cell lung cancer (NSCLC). So the aim of this study is to investigate the adjuvant role of CS in the treatment of NSCLC. The effects of the combination treatment of the polysaccharide-rich fraction of CS and cisplatin on H157 NSCLC cells were investigated through MTT assay for cell viability, lactate dehydrogenase and fluorescein diacetate and propidium iodide assay for cytotoxicity, and with flow cytometric analysis for apoptosis. The expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in H157 cells were detected by immunohistochemistry. Compared with the cells treated with cisplatin alone, cell viability was significantly decreased and the expression levels of VEGF and bFGF protein were significantly reduced in the cells treated with a combination of CS and cisplatin. The current study indicates that the polysaccharide of CS inhibits tumor growth in NSCLC and that CS may be a potential adjuvant chemotherapeutic agent in NSCLC therapy.

Examination of 12-lipoxygenase (12-LOX) as a therapeutic target in non-small cell lung cancer (NSCLC): Mechanisms controlling survival and induction of apoptosis following selective inhibition.

7068 Background: Platelet-type 12-LOX is an arachidonic acid metabolising enzyme resulting in the formation of 12(S)-HETE, which stimulates tumour cell adhesion, invasion and metastasis. This study aimed to examine the expression profile and role of this enzyme in NSCLC, and determine if it is a potential target for intervention. Methods: A panel of retrospective resected lung tumours was stained for 12-LOX expression by IHC. Levels of the 12-LOX metabolite, 12(S)-HETE, were examined in 50 NSCLC serum samples, and correlated with serum VEGF. A panel of NSCLC cell lines were treated with baicalein (10 uM), a selective inhibitor of 12-LOX, or 12(S)-HETE (100 ng/ml) and cell survival/proliferation examined by BrdU. Apoptosis following 12-LOX inhibition was examined by HCS and validated by FACS and DNA laddering. The effect of 12-LOX inhibition on NSCLC tumour growth and survival was examined in-vivo using an athymic nude mouse model. Gene alterations following 12-LOX inhibition in NSCLC cell lines were assessed by qPCR arrays and validated by RT-PCR. Transient transfection methods were used to examine the effects of 12-LOX overexpression in NSCLC cells. Results: 12-LOX expression was observed to a varying degree in human lung cancers of varying histological subtypes. 12(S)-HETE levels were correlated (p<0.05) with those of VEGF. Baicalein inhibited proliferation/survival in all cell lines, while 12(S)-HETE increased proliferation. 12-LOX inhibition increased apoptosis, indicated by a reduction in f-actin content and mitochondrial mass potential. Treatment with baicalein significantly reduced the growth of NSCLC tumours and increased overall survival in athymic nude mice. qPCR array data implicated a number of apoptosis/angiogenesis genes regulating these effects, including bcl-2, VEGF, integrin A2 and A4. 12-LOX overexpression resulted in an increase in VEGF secretion, confirming qPCR observations. Conclusions: 12-LOX is a survival factor/potential target in NSCLC, and correlated with angiogenic factor expression. Selective 12-LOX targeting may have clinical benefit for the future treatment.

Andrographolide down-regulates hypoxia-inducible factor-1α in human non-small cell lung cancer A549 cells

Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to inhibit non-small cell lung cancer (NSCLC) A549 cell migration and invasion via down-regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Here we demonstrated that Andro inhibited the expression of hypoxia-inducible factor-1α (HIF-1α) in A549 cells. HIF-1α plays an important role in tumor growth, angiogenesis and lymph node metastasis of NSCLC. The Andro-induced decrease of cellular protein level of HIF-1α was correlated with a rapid ubiquitin-dependent degradation of HIF-1α, and was accompanied by increased expressions of hydroxyl-HIF-1α and prolyl hydroxylase (PHD2), and a later decrease of vascular endothelial growth factor (VEGF) upon the treatment of Andro. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1α inactivation, because its DNA binding activity was suppressed by Andro. Molecular data showed that all these effects of Andro might be mediated via TGFβ1/PHD2/HIF-1α pathway, as demonstrated by the transfection of TGFβ1 overexpression vector and PHD2 siRNA, and the usage of a pharmacological MG132 inhibitor. Furthermore, we elucidated the involvement of Andro in HIF-1α transduced VEGF expression in A549 cells and other NSCLC cell lines. In conclusion, these results highlighted the potential effects of Andro, which may be developed as a chemotherapeutic or an anti-angiogenesis agent for NSCLC in the future.

Inhibition of Stat3 activation and tumor growth suppression of non-small cell lung cancer by G-quartet oligonucleotides

Lung cancer is the leading cause of cancer mortality in the United States. Despite advances made over the past decades, the overall survival of patients with lung cancer remains dismal. Here we report novel G-quartet oligodeoxynucleotides (GQ-ODN) that were designed to selectively target signal transducer and activator of transcription 3 (Stat3), in the treatment of human non-small cell lung cancer (NSCLC). The objective of this study was to evaluate the effects of two novel GQ-ODN STAT3 inhibitors, T40214 and T40231, on NSCLC bearing nude mice. NSCLC bearing nude mice were assigned to 5 groups, which were treated by vehicle, control ODN, T40214, T40231, and Paclitaxel, respectively. Tumors were measured, isolated and analyzed using Western blotting, immuno-histochemistry, RPA and TUNEL. Results show that GQ-ODN T40214 and T40231 significantly suppress the growth of NSCLC tumors in nude mice by selectively inhibiting the activation of Stat3 and its downstream proteins Bcl-2, Bcl-xL, Mcl-1, survivin, VEGF, Cyclin D1 and c-myc; thereby, promoting apoptosis and reducing angiogenesis and cell proliferation. These findings validate Stat3 as an important molecular target for NSCLC therapy and demonstrate the efficacy of GQ-ODN in inhibiting Stat3 phosphorylation.

Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer: A Review of Recent Clinical Trials

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related mortality in the United States. Therapeutic agents that target the underlying biology of this disease are necessary to improve outcomes. Angiogenesis plays a central role in NSCLC tumor growth and metastases. The vascular endothelial growth factor pathway (VEGF) as a therapeutic target was recently validated in NSCLC. Since then, a multitude of early phase clinical trials that incorporate the use of angiogenesis inhibitors, either as single agents or in combination with cytotoxic chemotherapy, have been conducted in advanced, refractory NSCLC. This article reviews these clinical trials with attention to toxicity, efficacy, and direction of further study. The data from these trials suggest that optimal use of anti-angiogenic agents in NSCLC is more likely in combination with standard cytotoxic agents, however the most effective combination with the least toxicity is yet to be determined.

Insulin-like growth factor-1 receptor/epidermal growth factor receptor (EGFR) heterodimerization and resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer

13032 Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been used to treat lung cancers, but resistance to these agents has been observed. This study was designed to investigate whether the insulin-like growth factor (IGF)-mediated signaling pathway induces resistance to the EGFR TKIs in lung cancer. Methods: The antitumor activities and action mechanisms of EGFR inhibitors (erlotinib, gefinitib, cetuximab), single or in combination with IGF-IR inhibitors, were assessed in vitro in a subset of non-small-cell lung cancer (NSCLC) cell lines by the MTT assay, flow cytometry-based TUNEL assay, soft agar, confocal microscopy, metabolic labeling, coimmunoprecipitation, and northern and western blot analyses, and in vivo in animal models. Correlation of EGFR and IGF-1R expression was assessed using lung tissues from patients with NSCLC. Results: EGFR TKI inhibited the proliferation and anchorage-dependent and -independent colony-forming ability of NSCLC cells, which induced apoptosis, only when IGF-1R signaling was blocked. Treatment with EGFR TKIs, but not with the EGFR antibody, induced EGFR:IGF-1R heterodimerization on cell surface and activation of the IGF-1R, resulting in the stimulation of PI3K/Akt/ mTOR pathway, promoting the de novo protein biosynthesis of survivin and EGFR, resulting in the survival of NSCLC cells. When IGF-IR and mTOR were blocked, treatment of EGFR-TKIs induced apoptosis in NSCLC cells, resulting in suppression of the NSCLC tumor growth. When we tested the expression of IGF-R and EGFR in human lung tissue, 9/14 tumor samples exhibited increased expression of EGFR and 7/9 samples showed a correlative increases in IGF-IR protein levels compared to their paired normal counterparts. Conclusions: These findings suggest that simultaneous targeting of EGFR and IGF-1R signaling pathways might be an effective therapeutic strategy against NSCLC. No significant financial relationships to disclose.

The 2005 Dolph Adams awards and the state of the Journal of Leukocyte Biology

The 2005 Dolph Adams awards and the state of the <i>Journal of Leukocyte Biology</i>

Radiotherapy Sensitization by Tumor-Specific TRAIL Gene Targeting Improves Survival of Mice Bearing Human Non–Small Cell Lung Cancer

To sensitize non-small cell lung cancer (NSCLC) to radiotherapy by tumor-specific delivery of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene. The TRAIL was delivered to human NSCLC cell lines and normal human bronchial epithelial cells by the replication-defective adenoviral vector Ad/TRAIL-F/RGD using a tumor-specific human telomerase reverse transcriptase promoter. Cancer growth was studied using 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt and clonogenic assays. Activation of the apoptosis pathway was analyzed in a Western blot and sub-G(1) DNA accumulation. A xenograft mouse lung cancer model was treated by intratumoral injections of Ad/TRAIL-F/RGD and local radiotherapy; the other groups received one of these treatments alone or a control agent. Apoptosis and TRAIL expression in tumors were also analyzed. Ad/TRAIL-F/RGD specifically targets human NSCLC cells without significant effect in normal human bronchial epithelial cells. The combination of Ad/TRAIL-F/RGD and radiotherapy significantly improved cell-killing effect in all NSCLC cell lines tested (P < 0.05). Expression of TRAIL showed a dose-dependent relationship with Ad/TRAIL-F/RGD, and radiation seemed to increase TRAIL expression. Activation of the apoptosis by TRAIL and radiation was shown by activation of caspase-9, caspase-8, caspase-3, and poly(ADP-ribose) polymerase and increased DNA sub-G(1) accumulation. The combination of TRAIL and radiotherapy significantly increased apoptosis in vivo, inhibited tumor growth, and prolonged mean survival in mice bearing human NSCLC to 43.7 days compared with 23.7 days (TRAIL only) and 16.5 days (radiotherapy only; P < 0.05). The combination of Ad/TRAIL-F/RGD and radiotherapy significantly improved therapeutic efficacy in suppressing NSCLC tumor growth and prolonging survival. Ad/TRAIL-F/RGD may improve the therapeutic ratio of radiotherapy in NSCLC.

Response of Non–Small Cell Lung Cancer Cells to the Inhibitors of Phosphatidylinositol 3-Kinase/Akt- and MAPK Kinase 4/c-Jun NH2-Terminal Kinase Pathways: An Effective Therapeutic Strategy for Lung Cancer

We previously showed that phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways cooperate to promote non-small cell lung cancer (NSCLC) cell proliferation in vitro. This study was designed to explore whether inhibition of these pathways effectively inhibits NSCLC tumor growth in vivo. The effects of PI3K/Akt inhibitors {LY294002, adenoviruses expressing dominant-negative mutant of the p85alpha adaptor subunit of PI3K (Ad-dnp85alpha), dominant-negative Akt [Ad-HA-Akt(KM)], or PTEN (Ad-PTEN)}, MKK4/c-jun NH2-terminal kinase (JNK) inhibitor [SP600215, adenovirus expressing dominant-negative MKK4, Ad-MKK4(KR)], and their combinations on proliferation and apoptosis in NSCLC cells were tested in vitro and in vivo using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, a flow cytometry-based terminal deoxynucleotidyl transferase-mediated nick-end labeling assay, Western blot and immunohistochemical analyses, and an NSCLC xenograft tumor model. Ad-dnp85alpha significantly inhibited proliferation of a subset of NSCLC cell lines used in our study. Intratumoral injection of Ad-dnp85alpha induced a significant decrease in the growth of H1299 NSCLC xenograft tumors. Concurrent inhibition of the PI3K/Akt and MKK4/JNK pathways showed enhanced antiproliferative effects on H1299 cells in vitro and in vivo by increasing apoptosis. PI3K/Akt and MKK4/JNK pathways cooperate to stimulate NSCLC cell proliferation by maintaining cell survival, suggesting that simultaneously targeting these two pathways might be an effective therapeutic strategy against NSCLC.

Interferon-gamma-inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases.

The success of solid tumor growth and metastasis is dependent upon angiogenesis. Neovascularization within the tumor is regulated, in part, by a dual and opposing system of angiogenic and angiostatic factors. We now report that IP-10, a recently described angiostatic factor, as a potent angiostatic factor that regulates non-small cell lung cancer (NSCLC)-derived angiogenesis, tumor growth, and spontaneous metastasis. We initially found significantly elevated levels of IP-10 in freshly isolated human NSCLC samples of squamous cell carcinoma (SCCA). In contrast, levels of IP-10 were equivalent in either normal lung tissue or adenocarcinoma specimens. The neoplastic cells in specimens of SCCA were the predominant cells that appeared to express IP-10 by immunolocalization. Neutralization of IP-10 in SCCA tumor specimens resulted in enhanced tumor-derived angiogenic activity. Using a model of human NSCLC tumorigenesis in SCID mice, we found that NSCLC tumor growth was inversely correlated with levels of plasma or tumor-associated IP-10. IP-10 in vitro functioned as neither an autocrine growth factor nor as an inhibitor of proliferation of the NSCLC cell lines. Reconstitution of intratumor IP-10 for a period of 8 wk resulted in a significant inhibition of tumor growth, tumor-associated angiogenic activity and neovascularization, and spontaneous lung metastases, whereas, neutralization of IP-10 for 10 wk augmented tumor growth. These findings support the notion that tumor-derived IP-10 is an important endogenous angiostatic factor in NSCLC.

Inhibition of interleukin-8 reduces tumorigenesis of human non-small cell lung cancer in SCID mice.

The salient feature of solid tumor growth is the strict dependence on local angiogenesis. We have previously demonstrated that IL-8 is an angiogenic factor present in freshly isolated specimens of human non-small cell lung cancer (NSCLC). Using a model of human NSCLC tumorigenesis in SCID mice, we now report that IL-8 acts as a promoter of human NSCLC tumor growth through its angiogenic properties. Passive immunization with neutralizing antibodies to IL-8 resulted in more than 40% reduction in tumor size and was associated with a decline in tumor-associated vascular density and angiogenic activity. IL-8 did not act as an autocrine growth factor for NSCLC proliferation. The reduction in primary tumor size in response to neutralizing antibodies to IL-8 was also accompanied by a trend toward a decrease in spontaneous metastasis to the lung. These data support the notion that IL-8 plays a significant role in mediating angiogenic activity during tumorigenesis of human NSCLC, thereby offering a potential target for immunotherapy against solid tumors.