Large Cell Carcinoma Cells Research Articles

MMP1 drives tumor progression in large cell carcinoma of the lung through fibroblast senescence.

Large cell carcinoma (LCC) is a rare and aggressive lung cancer subtype with poor prognosis and no targeted therapies. Tumor-associated fibroblasts (TAFs) derived from LCC tumors exhibit premature senescence, and coculture of pulmonary fibroblasts with LCC cell lines selectively induces fibroblast senescence, which in turn drives LCC cell growth and invasion. Here we identify MMP1 as overexpressed specifically in LCC cell lines, and we show that expression of MMP1 by LCC cells is necessary for induction of fibroblast senescence and consequent tumor promotion in both cell culture and mouse models. We also show that MMP1, in combination with TGF-β1, is sufficient to induce fibroblast senescence and consequent LCC promotion. Furthermore, we implicate PAR-1 and oxidative stress in MMP1/TGF-β1-induced TAF senescence. Our results establish an entirely new role for MMP1 in cancer, and support a novel therapeutic strategy in LCC based on targeting senescent TAFs.

Abstract 2021: Role of MMP1-PAR-1 crosstalk in the pro-tumorigenic senescent fibroblasts in large cell carcinoma of the lung

Abstract Tumor associated fibroblasts (TAFs) are key effector cells of cancer progression. Intriguingly, senescent TAFs have been reported in a growing list of agressive cancer subtypes including large cell carcinoma (LCC) of the lung. We previously reported that LCC cells induce fibroblast senescence in indirect co-cultures with transwells, revealing that paracrine signaling must be involved. A follow-up study identified MMP1 as an important regulatory protein in fibroblast senescence, since knocking-down MMP1 in LCC cells was sufficient to abrogate the induction of fibroblast senescence in co-cultures as well as the growth and invasion enhancement elicited by the conditioned medium of fibroblasts in LCC cells. Here we examined the potential role of the protease activated protein 1 (PAR-1) in fibroblast senescence by MMP1, since PAR-1 can be activated by MMP1 and has been reported in the stroma of lung cancer patients. We found that PAR-1 expression was down-regulated in fibroblasts that become senescent upon co-culture with LCC cells. In addition, forcing a reduced PAR-1 expression in fibroblasts by siRNA increased their expression of markers of the senescence associated secretory phenotype (SASP) during co-culture with LCC cells, whereas the percentage of senescence-associated beta-galactosidase positive fibroblasts remained largely unaffected. These results suggest that fibroblast senescence elicited by MMP1 secreted by LCC cells is mediated by processes other than PAR-1 activation and/or overexpression in fibroblasts. Citation Format: Marta Gabasa, Rafael Ikemori, Evette Radisky, Noemí Reguart, Derek Radisky, Jordi Alcaraz. Role of MMP1-PAR-1 crosstalk in the pro-tumorigenic senescent fibroblasts in large cell carcinoma of the lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2021.

Chinese native medicinal plant Euphorbiaceae show antitumor and anti-oxidant features in lewis lung cancer-bearing mice

Background: As a promising means, natural products from Chinese herb provide valuable sources for cancer therapy. Euphorbiaceae has been used as a remedy for the treatment of many diseases, including cancer. However, studies about its effects on lung cancer are limited, and the mechanisms are not well established. Objective: The present study aims to investigate the anticancer effects of Euphorbiaceae extract (EE) in vitro and in vivo, as well as the potential mechanisms. Materials and Methods: In the present study, 3-(4,5-dimethythiazol-. 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to investigate the effects of EE on Lewis lung adenocarcinoma cell (LLC) viability. Flow cytometric analysis was used to observe the changes of apoptosis and cell cycle of large cell carcinoma (LCC). The gene expressions were detected by real-time reverse transcription–polymerase chain reaction and Western blot. The tumorigenicity assay was performed to evaluate the inhibitory effects of EE in vivo. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were also determined. Results: MTT assay demonstrated that EE significantly inhibited the growth of LLC in a dose-dependent manner in vitro. Flow cytometric analysis revealed that EE markedly induced apoptosis and G0/G1 arrest of the LCC cells. Furthermore, we found that EE led to the expression changes of apoptosis-related genes, with the decrease of Bcl-2 and the increase of Bax and caspase-9. The results from tumor-bearing animals further confirmed that the administration of EE significantly suppressed the tumor growth in vivo. Meanwhile, the activities of serum SOD, CAT, and GSH-Px increased significantly after the treatment of EE. Conclusion: These results suggested that the anticancer effects of EE may be involved in apoptosis induction, proliferation suppression, and oxidant scavenging.

Litchi seed extract inhibits epidermal growth factor receptor signaling and growth of Two Non-small cell lung carcinoma cells

BackgroundLitchi seeds possess rich amounts of phenolics and have been shown to inhibit proliferation of several types of cancer cells. However, the suppression of EGFR signaling in non-small cell lung cancer (NSCLC) by litchi seed extract (LCSE) has not been fully understood.MethodsIn this study, the effects of LCSE on EGFR signaling, cell proliferation, the cell cycle and apoptosis in A549 adenocarcinoma cells and NCI- H661 large-cell carcinoma cells were examined.ResultsThe results demonstrated that LCSE potently reduced the number of cancer cells and induced growth inhibition, cell-cycle arrest in the G1 or G2/M phase, and apoptotic death in the cellular experiment. Only low cytotoxicity effect was noted in normal lung MRC-5 cells. LCSE also suppressed cyclins and Bcl-2 and elevated Kip1/p27, Bax and caspase 8, 9 and 3 activities, which are closely associated with the downregulation of EGFR and its downstream Akt and Erk-1/-2 signaling.ConclusionThe results implied that LCSE suppressed EGFR signaling and inhibited NSCLC cell growth. This study provided in vitro evidence that LCSE could serve as a potential agent for the adjuvant treatment of NSCLC.

Lung Cancer-Targeting Peptides with Multi-subtype Indication for Combinational Drug Delivery and Molecular Imaging.

Lung cancer is the leading cause of cancer-related death worldwide. Most targeted drugs approved for lung cancer treatment are tyrosine kinase inhibitors (TKIs) directed against EGFR or ALK, and are used mainly for adenocarcinoma. At present, there is no effective or tailored targeting agent for large cell carcinoma (LCC) or small cell lung cancer (SCLC). Therefore, we aimed to identify targeting peptides with diagnostic and therapeutic utility that possess broad subtype specificity for SCLC and non-small cell lung cancer (NSCLC). We performed phage display biopanning of H460 LCC cells to select broad-spectrum lung cancer-binding peptides, since LCC has recently been categorized as an undifferentiated tumor type within other histological subcategories of lung cancer. Three targeting phages (HPC1, HPC2, and HPC4) and their respective displayed peptides (HSP1, HSP2, and HSP4) were able to bind to both SCLC and NSCLC cell lines, as well as clinical specimens, but not to normal pneumonic tissues. In vivo optical imaging of phage homing and magnetic resonance imaging (MRI) of peptide-SPIONs revealed that HSP1 was the most favorable probe for multimodal molecular imaging. Using HSP1-SPION, the T2-weighted MR signal of H460 xenografts was decreased up to 42%. In contrast to the tight binding of HSP1 to cancer cell surfaces, HSP4 was preferentially endocytosed and intracellular drug delivery was thereby effected, significantly improving the therapeutic index of liposomal drug in vivo. Liposomal doxorubicin (LD) conjugated to HSP1, HSP2, or HSP4 had significantly greater therapeutic efficacy than non-targeting liposomal drugs in NSCLC (H460 and H1993) animal models. Combined therapy with an HSP4-conjugated stable formulation of liposomal vinorelbine (sLV) further improved median overall survival (131 vs. 84 days; P = 0.0248), even in aggressive A549 orthotopic models. Overall, these peptides have the potential to guide a wide variety of tailored theranostic agents for targeting therapeutics, non-invasive imaging, or clinical detection of SCLC and NSCLC.

MA17.09 Premature Fibroblast Senescence in Large Cell Carcinoma Provides Enhanced Growth and Invasive Advantages to Cancer Cells in Culture and in vivo

Tumor-associated fibroblasts (TAFs) are increasingly regarded as essential co-conspirators for tumor progression in all solid tumors including non-small cell lung cancer. While most TAFs exhibit activation markers indicative of a myofibroblast-like phenotype, senescence markers have been reported in a growing list of selected cancer types only. However, the presence of senescent TAFs in lung cancer remains undefined. Assessing senescence in lung TAFs is important because previous studies have reported that senescent TAFs enhances tumor growth, which is in marked contrast with the widely accepted tumor-suppressive role of senescence in cancer cells. We examined common senescence markers in patient derived lung TAFs from the 3 major non-small cell lung cancer (NSCLC) subtypes: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Given the difficulties in gathering LCC-TAFs owing to the lower prevalence of LCC compared to the other subtypes, primary fibroblasts from 2 independent fibroblast collections were used. Senescence markers included senescence-associated beta-galactosidase, permanent growth arrest and spreading. We found an enrichment of the myofibroblast-like phenotype in TAFs regardless their histologic subtype, yet senescence was observed in LCC-TAFs only regardless their neuroendocrine status. Likewise, co-culturing normal lung fibroblasts with LCC (but not ADC or SCC) cancer cells was sufficient to induce senescence, and this induction was prevented in the presence of an antioxidant, indicating that it is mediated through oxidative stress. Remarkably, senescent fibroblasts provided growth and invasive advantages to LCC cells in culture and in vivo beyond those effects provided by control (non-senescent) fibroblasts. Our findings expand recent evidence that challenges the common assumption that lung TAFs are a heterogeneous myofibroblast-like cell population regardless their histologic subtype. Of note, because LCC often distinguishes itself in the clinic by its aggressive nature, our findings support that senescent or senescent-like TAFs may contribute to the selective aggressive behavior of LCC tumors.

Abstract 4103: Cancer cell-stromal cell crosstalk drives fibroblast senescence and tumor progression in large cell carcinoma of the lung in culture and in vivo

Abstract Permanent cell cycle arrest through senescence has been previously regarded as a protective mechanism against tumor progression. In contrast, there is evidence that senescence in tumor-associated fibroblasts (TAFs) enhances tumor growth. Senescence has been reported in tumor associated fibroblasts (TAFs) from a growing list of selected cancer types. However, the presence of senescent TAFs in lung cancer remains undefined. To address this gap of knowledge, we examined common markers of senescence in primary TAFs from the 3 major non-small cell lung cancer (NSCLC) subtypes: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Given the difficulties in gathering LCC-TAFs owing to the lower prevalence of LCC compared to the other subtypes, primary fibroblasts from 2 independent cell collections were used. We found an enrichment of the myofibroblast-like phenotype in TAFs regardless their histologic subtype, yet senescence was observed in LCC-TAFs only. Likewise, co-culturing normal lung fibroblasts with LCC (but not ADC or SCC) cancer cells was sufficient to induce senescence, and this induction was prevented in the presence of an antioxidant, indicating that it is mediated through oxidative stress. Of note, senescent fibroblasts provided growth and invasive advantages to LCC cells in culture and in vivo beyond those provided by control (non-senescent) fibroblasts. These results expand recent evidence that challenges the common assumption that lung TAFs are a heterogeneous myofibroblast-like cell population regardless their histologic subtype. Of note, because LCC often distinguishes itself in the clinic by its aggressive nature, our findings support that senescent or senescent-like TAFs may contribute to the selective aggressive behavior of LCC tumors. Citation Format: Roberto Lugo, Marta Gabasa, Francesca Andriani, Marta Puig, Federica Facchinetti, Josep Ramírez, Abel Gómez-Caro, Ugo Pastorino, Pere Gascón, Albert Davalos, Noemí Reguart, Luca Roz, Jordi Alcaraz. Cancer cell-stromal cell crosstalk drives fibroblast senescence and tumor progression in large cell carcinoma of the lung in culture and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4103.

Heterotypic paracrine signaling drives fibroblast senescence and tumor progression of large cell carcinoma of the lung.

Senescence in cancer cells acts as a tumor suppressor, whereas in fibroblasts enhances tumor growth. Senescence has been reported in tumor associated fibroblasts (TAFs) from a growing list of cancer subtypes. However, the presence of senescent TAFs in lung cancer remains undefined. We examined senescence in TAFs from primary lung cancer and paired control fibroblasts from unaffected tissue in three major histologic subtypes: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Three independent senescence markers (senescence-associated beta-galactosidase, permanent growth arrest and spreading) were consistently observed in cultured LCC-TAFs only, revealing a selective premature senescence. Intriguingly, SCC-TAFs exhibited a poor growth response in the absence of senescence markers, indicating a dysfunctional phenotype rather than senescence. Co-culturing normal fibroblasts with LCC (but not ADC or SCC) cancer cells was sufficient to render fibroblasts senescent through oxidative stress, indicating that senescence in LCC-TAFs is driven by heterotypic signaling. In addition, senescent fibroblasts provided selective growth and invasive advantages to LCC cells in culture compared to normal fibroblasts. Likewise, senescent fibroblasts enhanced tumor growth and lung dissemination of tumor cells when co-injected with LCC cells in nude mice beyond the effects induced by control fibroblasts. These results define the subtype-specific aberrant phenotypes of lung TAFs, thereby challenging the common assumption that lung TAFs are a heterogeneous myofibroblast-like cell population regardless of their subtype. Importantly, because LCC often distinguishes itself in the clinic by its aggressive nature, we argue that senescent TAFs may contribute to the selective aggressive behavior of LCC tumors.

Differential effects of MTSS1 on invasion and proliferation in subtypes of non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) accounts for >80% of all cases of lung cancer and can be divided into lung adenocarcinoma (LAC), large-cell carcinoma (LCC), and squamous cell carcinoma (SCC). Accumulating evidence suggests that MTSS1, which is a newly discovered protein associated with tumor progression and metastasis, may have differential roles in cancer malignancy. As it has been demonstrated that MTSS1 is overexpressed in NSCLC and may be an independent prognostic factor in patients with SCC, the present study explored the differential roles of MTSS1 in the invasion and proliferation of different subtypes of NSCLC. Stable overexpression and knockdown of MTSS1 was performed in human NSCLC H920 (LAC), H1581 (LCC) and SW900 cell lines (SCC), and western blot, cell invasion, proliferation and FAK activity analyses were used to investigate the effects. Overexpression of MTSS1 enhanced the invasion and proliferation abilities of H920 and H1581 cells, and these effects were abolished by treatment with selective FAK inhibitor 14, which did not affect the expression of MTSS1. Notably, overexpression of MTSS1 inhibited invasion and proliferation in SW900 cells, and this effect was enhanced by the selective FAK inhibitor. Knockdown of MTSS1 decreased the invasion and proliferation abilities of H920 and H1581 cells, whereas knockdown increased invasion and proliferation in SW900 cells. Furthermore, while overexpression of MTSS1 induced FAK phosphorylation and activity in H920 and H1581 cells, MTSS1 overexpression inhibited FAK phosphorylation/activity in SW900 cells. Knockdown of MTSS1 decreased FAK phosphorylation/activity in H920 and H1581 cells, whereas knockdown increased these processes in SW900 cells. To the best of our knowledge, the present study was the first to demonstrate that MTSS1 has differential roles in various subtypes of NSCLC, acting via a FAK-dependent mechanism. The results indicated that MTSS1 may enhance invasion and proliferation in LAC and LCC cells, whereas MTS11 inhibits these processes in SCC cells. These findings provide novel insight into the functional role of MTSS1 in cancer and may help elucidate therapeutic strategies for the treatment of various types of cancer.

Upregulation of MMP-2 by HMGA1 Promotes Transformation in Undifferentiated, Large-Cell Lung Cancer

Although lung cancer is the leading cause of cancer death worldwide, the precise molecular mechanisms that give rise to lung cancer are incompletely understood. Here, we show that HMGA1 is an important oncogene that drives transformation in undifferentiated, large-cell carcinoma. First, we show that the HMGA1 gene is overexpressed in lung cancer cell lines and primary human lung tumors. Forced overexpression of HMGA1 induces a transformed phenotype with anchorage-independent cell growth in cultured lung cells derived from normal tissue. Conversely, inhibiting HMGA1 expression blocks anchorage-independent cell growth in the H1299 metastatic, undifferentiated, large-cell human lung carcinoma cells. We also show that the matrix metalloproteinase-2 (MMP-2) gene is a downstream target upregulated by HMGA1 in large-cell carcinoma cells. In chromatin immunoprecipitation experiments, HMGA1 binds directly to the MMP-2 promoter in vivo in large-cell lung cancer cells, but not in squamous cell carcinoma cells. In large-cell carcinoma cell lines, there is a significant, positive correlation between HMGA1 and MMP-2 mRNA. Moreover, interfering with MMP-2 expression blocks anchorage-independent cell growth in H1299 large-cell carcinoma cells, indicating that the HMGA1-MMP-2 pathway is required for this transformation phenotype in these cells. Blocking MMP-2 expression also inhibits migration and invasion in the H1299 large-cell carcinoma cells. Our findings suggest an important role for MMP-2 in transformation mediated by HMGA1 in large-cell, undifferentiated lung carcinoma and support the development of strategies to target this pathway in selected tumors.

The effect of thalidomide on non-small cell lung cancer (NSCLC) cell lines: possible involvement in the PPARgamma pathway.

Lung cancer is the leading cause of cancer-related death in developed countries. Non-small cell lung cancer (NSCLC) represents 80% of the total lung cancer cases and is comprised of adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma and large cell carcinoma (LCC) subtypes. The ability of LCC to metastasize earlier than the other forms of lung cancer suggests anti-angiogenic drugs as effective agents to combat this cancer. Thalidomide is an anti-angiogenic drug that has shown promise in multiple hematological diseases, and myeloma and other cancers. However, the molecular mechanism by which thalidomide exerts its effects is poorly understood. Therefore, we evaluated the effectiveness of thalidomide on NSCLC cell growth, and found that LCC cells were growth inhibited by 40-60%. This effect seemed specific to LCC cancer cells, since other forms of NSCLC were only mildly affected by thalidomide. At the molecular level, thalidomide increased peroxisome proliferator-activated receptor gamma (PPARgamma) protein dose-dependently, and peroxisome proliferator response element activity. Further, thalidomide treatment of LCC cells decreased nuclear factor kappa B activity in a dose-dependent fashion, increased apoptosis and decreased the expression of angiogenic proteins. In our mouse xenograft model of lung cancer, we found that intratumoral thalidomide caused a 64% decrease in tumor growth; moreover, tumors from the thalidomide-treated mice expressed higher PPARgamma, than tumors from control mice. This study shows the antitumor activity of thalidomide against LCC tumors and suggests a model in which thalidomide exerts its antitumor effects on LCC cells through the induction of PPARgamma and subsequent downstream signaling. To our knowledge, this is the first study to show a link between thalidomide and PPARgamma.

Establishment and characterization of cell lines from human small cell and large cell carcinomas of the lung.

Five new small cell carcinomas (SCC) cell lines and a large cell carcinoma (LCC) cell line were established from human lung cancers. The SCC cell lines had, as a group, common phenotypic properties which distinguished them from non-SCC cell lines. However, the studies also revealed a considerable biological heterogeneity among the individual SCC cell lines. Thus, the SCC cell lines had a typical growth pattern with cell clusters in suspension or partly adherent to the bottom. All the lines examined grew in agarose with variable cloning efficiencies, and all but one line formed tumors subcutaneously in nude mice. The ultrastructure of the SCC cell lines was characteristic with dense core granules at a variable frequency. Neuron-specific enolase was detectable in all SCC cell lines, usually in large amounts, and an inconstant production of a spectrum of polypeptide hormones was found, typical of SCC. The LCC cells proliferated in monolayers, formed colonies in agarose and grew in nude mice. Ultrastructurally, the LCC cell line differed from the SCC cell lines in having intra- and intercellular lumina and tonofilaments. The capacity of the LCC and a previously established squamous cell carcinoma cell line (U-1752) to produce neuron-specific enolase and polypeptide hormones was characteristically much lower than that of the SCC cell lines. We conclude from this study that SCC cell lines, although individually distinct from one another, are quite homogeneous as a group in expressing a set of basic common neuro-endocrine markers. However, these studies also suggest some biological relationship between SCC, LCC and SQC by virtue of their expression of some common neuro-endocrine markers, in support of the concept of a common histogenetic origin of human lung cancers.