Abstract 1216: Effects of phenformin in LKB1/KRAS mutated non-small cell lung cancer patient derived xenograft

Abstract Metformin is an oral antidiabetic drug of the biguanide class. Several epidemiological and case-control studies reported that diabetics using metformin may have lower cancer risk in comparison to those using other antidiabetic medications. Recent studies in experimental models suggest that another biguanide, phenformin, has higher anticancer activity compared with metformin. The aim of this study was to evaluate the effects of metformin and phenformin in Non-Small Cell Lung Cancer (NSCLC) models. In particular, the effects of these compounds in LKB1 or LKB1/KRAS mutated compared to wild type models have been investigated. NSCLC cell lines (A549, H460, H1299, LT73) have been treated with either metformin or phenformin to evaluate in vitro dose/response depending on the different mutational status of KRAS and LKB1 genes. The effects of the two biguanide have been investigated also in vivo models, exploiting the panel of Patient Derived Xenografts (PDXs) directly generated from NSCLC patients in our Institute. In vitro experiments indicated that cell lines with already impaired metabolism (LKB1 and/or KRAS mutated) were more sensitive to phenformin treatment. Interestingly, treatments of these cell lines with r phenformin resulted in a 50% decrease in the amount of CD133+ Tumor Initiating Cells (TIC). Moreover, high doses of phenformin (LD75) did not result in the increase of TIC previously observed after Cisplatin treatment. In vivo, early treatments (Tumor Volume = 50-100mm3) of an LKB1 mutated PDX model resulted in a significant reduction of tumor volume (300 ± 50 mm3 in phenformin treated vs 750 ± 100 mm3 in vehicle treated tumors) already after three weeks of treatment (100mg/Kg/day). Importantly, no regrowth was observed in responding tumors even after the end of treatments. No effects were observed in LKB1 wild type PDX treated with phenformin. Taken together, these preliminary data indicate a selective action of phenformin on LKB1 mutated lung adenocarcinoma models in vitro and in vivo. Interestingly, this compound seems to be more active in TIC than normal chemotherapeutic drugs (i.e. cisplatin). Effects of biguanide on TIC could be the cause of the lack of tumor regrowth after treatment, thus making these compounds potentially useful in counteracting disease recurrence and/or metastasis formation. Further investigations are needed to elucidate the different effects of metformin and phenformin in double mutated (LKB1 and KRAS) PDXs models. Citation Format: Massimo Moro, Luca Roz, Roberto Caserini, Ugo Pastorino, Gabriella Sozzi. Effects of phenformin in LKB1/KRAS mutated non-small cell lung cancer patient derived xenograft. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1216. doi:10.1158/1538-7445.AM2014-1216</jats:p

Physical limits to Economic Growth

The debate on the physical limits and constraints to the economic growth of globalized society is now widespread. This book explores the physical and economic aspects of the conflict between humans, with their thoughtless focus on growth through material production, and environmental constraints. In the context of the looming shortage of material resources and the latest science on climate change, Physical Limits to Economic Growth offers new insights which provide a broad and comprehensive picture of the conflict between humans and environmental constraints. The authors’ approach goes beyond the boundaries of specialized disciplines to explore climate change, resource depletion, technical innovation and the interactions between these within the socio-economic-institutional systems we live in. This volume looks at opportunities for rethinking these systems if we moved away from fossil fuel dependence, while considering the status of current mainstream economic thinking around this subject. Physical Limits to Economic Growth provides a genuine interdisciplinary examination of the physical limits to economic growth. It will be of interest to both students and academics in various disciplines in the areas of natural sciences, climate change and economics

Abstract 3898: Disseminated cells from primary lung cancers contain a distinct cancer initiating subpopulation with mesenchymal-like features

Abstract Cancer initiating cells (CICs) have been shown to be responsible for primary tumor formation, drug resistance and metastatic dissemination. We previously demonstrated that the subset of CD133+ lung CICs co-expressing the chemokine receptor CXCR4, is resistant to chemotherapy and endowed with high metastasis initiating ability using in vivo experimental metastasis assay. To investigate the role of the subset of CD133+CXCR4+ cells in the in vivo spontaneous tumor dissemination process, we analyzed lungs of mice bearing subcutaneous implants of lung cancer patients derived xenografts (PDXs). Using FACS and Real Time PCR analysis a small fraction of lung disseminated tumor cells (DTCs: 0.001% to 0.01%) was detected in 9/11 different PDX models, likely remaining at single cells state as suggested by the lack of histologically apparent metastasis. In particular, compared to parental subcutaneous tumors, lung DTCs were enriched for CD133+CXCR4+ CICs not expressing the epithelial antigen EpCAM. Modifying an innovative cancer cells culture method that allows the recovery of highly purified and viable tumor cells (Kondo J. et al. 2011, PNAS), we also generated murine lung tissue spheroids that were 100 fold enriched in DTCs. Real Time PCR analysis of lung spheroids consistently revealed an up-regulation of stemness and EMT-associated genes compared to the parental tumor, in accordance with the enrichment for disseminating CD133+CXCR4+EpCAM- CICs subset observed by FACS. Subcutaneous injection in SCID mice of lung spheroids containing approximately 200 DTCs, generated tumors resembling the original PDX, but also showing an increased fraction of CD133+ stem like cells and particularly of the CD133+CXCR4+EpCAM- subset. Moreover, cells recovered from DTCs-originated tumors were able to grow in vitro as spheres in anchorage independent condition, up-regulated EMT-genes and showed an enhanced tumorigenic potential and increased ability to disseminate to murine lung compared with original PDX in in vivo serial transplantation assay, indicating that the fraction of CD133+CXCR4+EpCAM- CICs enriched in DTCs was able to efficiently originate tumors with distinctive traits. We also found that the fraction of CD133+ cells and particularly the CD133+CXCR4+EpCAM- subset were increased in patient's metastatic lymph nodes compared to primary tumors, confirming in a clinical setting the role of CD133+CXCR4+EpCAM- cells as metastasis initiating cells. Our data functionally proves that lung DTCs contain a specific subset of tumor initiating cells that is able to generate tumors showing distinct properties and enrichment in the mesenchymal-like CICs compartment. The relevance of this finding is confirmed in clinical setting, identifying a new possible target for novel therapies aimed at interfering with metastatic dissemination, which represents the main reason for lung cancer poor outcome.. Citation Format: Giulia Bertolini, Massimo Moro, Monica Tortoreto, Roberto Caserini, Ugo Pastorino, Luca Roz, Gabriella Sozzi. Disseminated cells from primary lung cancers contain a distinct cancer initiating subpopulation with mesenchymal-like features. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3898. doi:10.1158/1538-7445.AM2014-3898</jats:p

Inactivation of Both FHIT and p53 Cooperate in Deregulating Proliferation-Related Pathways in Lung Cancer

IntroductionFHIT and p53 are the two most commonly altered tumor suppressor genes in lung cancer, and their molecular status regulates sensitivity to anticancer drugs. Although their functions are independent, there is evidence that their pathways might be interconnected, but little is known at the molecular level.MethodsMicroarray profiling of FHIT-transduced lung cancer cells and modulation of FHIT levels by RNA interference in human bronchial cells were used to generate a signature of FHIT-regulated transcripts. Expression of these genes was evaluated by real-time polymerase chain reaction in 55 primary lung cancer samples characterized for FHIT and p53 expression by immunehistochemistry.ResultsA signature of FHIT-transcripts, particularly enriched in genes involved in cell cycle control, was identified. This signature showed overlap with p53-regulated genes, indicating possible crosstalk between these proteins. Consistently, transcriptional deregulation after FHIT modulation was higher in p53-negative cells. In primary lung cancers, inactivation of either gene was detected in 48 of 55 cases (87%) and both genes in 23 of 55 (42%) cases, confirming the central role of these pathways. Primary tumors with inactivation of both FHIT and p53 displayed the strongest deregulation of growth-related pathways with high levels of expression of CCNB1, BUB1, CDC6, TOP2A, MCM6, and CENPF.ConclusionsFHIT and p53 seem to rely on common mediators, and inactivation of both genes results in prominent deregulation of growth-related pathways in lung cancer cell lines and primary tumors. This reveals crosstalk between these proteins and suggests a possible distinctive phenotype for tumors with inactivation of both genes

Abstract 1483: Lung derived fibroblasts influence extracellular matrix composition and dissemination of lung cancers

Abstract There is increasing evidence of the central contribution of tumor microenvironment to cancer development and progression. The elucidation of the role of different cellular components to this mechanism might therefore provide novel insights on the transformation process and potentially indicate targets for innovative therapeutic intervention. In particular, it has been shown that fibroblasts isolated from invasive breast lesions can contribute to tumor growth through the CXCR4/SDF1 pathway and that human prostatic carcinoma-associated fibroblasts (CAFs) can drive the progression of initiated non-tumorigenic epithelial cells. Recently a gene-expression signature derived from lung CAFs was shown to have prognostic value in non-small cell lung cancer (NSCLC) patients. To further investigate the contribution of fibroblasts to lung cancer we established primary cultures of CAFs and matched normal fibroblasts (NFs) from resected NSCLC and performed co-injection experiments in nude mice with A549 lung cancer cells. Co-injection of fibroblasts generally resulted in tumors of bigger size and/or increased take rate when cells were injected at low doses. Time-course experiments revealed presence of human cells in the stromal component of xenografts up to one month after injection, indicating possible involvement also in the progression phase. To evaluate the contribution of fibroblasts to extracellular matrix composition of tumors we therefore analyzed by real-time PCR with human specific primers the expression levels of 14 extracellular matrix (ECM) related genes in 63 tumors generated by co-injection of lung cancer cells and fibroblasts (CAFs or NFs). Compared to tumors generated by injection of A549 cells alone, heterotypic tumors displayed strongly increased levels of COL6A3 and MMP2 (p≤0.03), slightly increased levels of SPARC and reduced CTSL and CTSC (p≤0.01) indicating influence of fibroblasts on ECM composition. Tumors generated by co-injections were also more likely to metastasize to the lungs as determined by flow cytometry and real-time PCR (31% vs. 17%; OR=2.56 95% CI 0.6-12.7). Moreover culturing of primary lung cancer cells with CAF conditioned medium (CM) also resulted in similar transcriptional regulations of ECM-related genes and increased levels of MMP2 were detected in tumors derived from injection of CM treated cells. Taken together these data demonstrate that cross-talk between fibroblasts and cancer cells can dictate ECM composition and regulate dissemination of lung cancer and suggest that identification of factors responsible for this cross-talk could be instrumental in devising novel therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1483. doi:1538-7445.AM2012-1483</jats:p

Abstract 530: Contribution of fibroblasts isolated from primary lung cancers and normal lung tissue to tumor growth

Abstract Fibroblasts isolated from cancer specimens have been shown to possess pro-tumorigenic properties, often associated with traits reminiscent of myofibroblasts found at sites of wound healing, but little is known about the mediators of the interaction between fibroblasts and cancer cells especially in lung cancer. To evaluate the contribution of fibroblasts to lung cancer development we established 60 fibroblast cultures from cancerous and normal lung tissue from primary lung cancer patients, including 18 pairs of normal (NF) and cancer-associated fibroblasts (CAF) from the same patient. The mesenchymal nature of the cultures was confirmed by expression of surface markers CD90, CD166, CD105 and CD73 and negativity for epithelial and hematopoietic markers (ESA and CD45). Further immunophenotyping with markers specific for fibroblasts subpopulations (FSP, vimentin, alpha-SMA, FAP) identified frequent expression of activation markers, with up to 50% of alpha-SMA+ cells. Interestingly activation properties were identified also in cultures derived from normal lung tissue. Co-mingling of fibroblasts (NF or CAF) with A549 lung cancer cells generally resulted in increased tumor size after injection in nude mice and 2/4 of the CAF cultures tested also increased tumor take when cancer cells were injected at low doses. Human cells were identified by HLA staining in the stroma of tumors grown after co-injection confirming the contribution of the mixed fibroblasts to tumor development. Furthermore medium conditioned by fibroblasts promoted growth of lung cancer cells or immortalized bronchial epithelial cells. All the cultures tested displayed a normal karyotype suggesting that the functional properties of fibroblasts are likely to be the result of epigenetic changes.These results indicate that lung fibroblasts might play a central role in the growth and progression of lung neoplasia. Consistently with their activated phenotype also cultures from normal lung tissue of heavy smokers displayed pro-tumorigenic activity, indicating that the microenvironment of lungs heavily exposed to damage from carcinogenic substances might already be predisposed to foster the growth of transformed epithelial cells. The full dissection of this complex picture might reveal mediators of the cross-talk between stroma and cancer cells and provide novel therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 530. doi:10.1158/1538-7445.AM2011-530</jats:p

Abstract 4867: Microenvironment stimuli elicited by fibroblasts contribute to epithelial mesenchymal transition and acquisition of stemness phenotype in lung cancer

Abstract Epithelial cancers could be described as abnormal organs composed of cells with different phenotype and functional properties. It has been suggested that only a small fraction of cells defined as “cancer stem cells” (CSCs) is capable of initiating and maintaining a new tumor and that local microenvironment could be involved in modulation of stemness properties and metastatic colonization by induction of the epithelial-mesenchymal transition (EMT). To investigate how signals from stromal fibroblasts can influence stemness properties we used a primary lung adenocarcinoma cell line (LT73) containing a subpopulation of CD133+ CSCs (0.1%) and its derivative devoid of CD133+ cells (LT73CD133neg) which remains stable during short-term culturing. Medium conditioned by primary fibroblasts cell lines (CM) resulted in increase of CD133 positive cells, more pronounced in LT73CD133neg, indicating positive regulation of the stem cell pool and de novo generation of CSCs. Accordingly, expression of stemness related genes OCT4, NANOG and GA6 was increased (fold change 1.5-3). In vivo experiments also confirmed that tumors generated by injection of CM-treated cells had greater size compared to controls and maintained increased stemness features. Moreover stimulation with CM, associated to generation of CD133+ cells, was also often accompanied by gene expression changes consistent to EMT activation. To verify the importance of fibroblasts in providing the right cues also for successful colonization of the lungs, LT73 cells were injected in the tail vein of SCID mice. After 2 months LT73 cells co-injected with fibroblasts were present in the lungs in larger numbers (4 fold increase) compared to control mice indicating a possible role for fibroblasts in the preparation of the pre-metastatic niche Finally to establish whether the EMT process was required for acquisition of a stem like phenotype in presence of microenvironmental stimuli, we overexpressed in LT73 wt cell line miR200c which has been shown to prevent TGFbeta-induced EMT. Preliminary experiments showed that, although miR200c was able to partially prevent the EMT process by contrasting downregulation of the epithelial marker CDH1, its overexpression was insufficient to abrogate upregulation of the mesenchymal markers SNAI2, VIM, FN1 and de novo generation of stem-like cells (CD133+) after TGFbeta treatment. This indicates that even partial activation of the EMT process could be sufficient to modulate stemness features. Together these data demonstrate that stimuli from fibroblasts could de novo generate CD133+ cells modulating stem cells phenotype probably through EMT process and that the proficient cross-talk between fibroblasts and cancer stem cells could also be relevant for metastatic colonization. Citation Format: Francesca Andriani, Tiziana Caputo, Giulia Bertolini, Federica Facchinetti, Erika Baldoli, Massimo Moro, Roberto Caserini, Gabriella Sozzi, Luca Roz. Microenvironment stimuli elicited by fibroblasts contribute to epithelial mesenchymal transition and acquisition of stemness phenotype in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4867. doi:10.1158/1538-7445.AM2014-4867</jats:p

Abstract 1395: Conversion to stem cell state in response to microenvironmental cues is regulated by balance between epithelial and mesenchymal features in lung cancer

Abstract Epithelial cancers consist in heterogeneous populations of cells characterized by different phenotypic and biological features. Neoplastic progression is driven by a subpopulation of cells within the tumor bulk defined as “cancer initiating cells”(CICs) with stemness features. Modulation of cancer initiating properties is a consequence of genetic and epigenetic changes in cancer cells combined with their interaction with the surrounding microenvironment. To investigate how microenvironmental signals could influence the stemness properties and how these signals are integrated in tumors with different phenotypic features, eight non-small cell lung cancer cell lines were exposed to external stimuli such as TGFβ. The frequency of CD133, previously identified as a marker of stemness in lung cancer, was evaluated by flow cytometry and the expression of epithelial and mesenchymal genes was analyzed by Real-Time PCR and immunofluorescence (IF) or immunohystochemistry (IHC). TGFβ treatment resulted in heterogeneous increased of CD133+ cells and the extent of response was strictly dependent on the ratio between epithelial and mesenchymal features, estimated by the expression of E-cadherin and SNAI2 genes. Only cells that reached a critical threshold in the balance between epithelial and mesenchymal traits were able to generate CICs, acquiring increased in vivo tumorigenic properties in response to external stimuli. Consistently, in cell lines that reached the critical threshold by modulating the epithelial-mesenchymal balance through over expression of miR-200c or demethylation of E-cadherin promoter, the ability to acquire stemness phenotype under microenvironmental stimuli was restored. Single-cell analysis showed the presence of a sub-population of cells expressing both epithelial and mesenchymal markers that could be responsible of the phenotypic plasticity within different cell lines. Moreover, the evaluation of 60 primary lung tumors by IHC discovered a specific pattern of E-cadherin a SNAI2 that identify patients with worst prognosis. Together these findings provided new insight to understand how dynamic modulation of epithelial and mesenchymal properties determines the plasticity of lung cancer cells through the generation of cancer initiating cells in response to microenviromental stimuli. Citation Format: Francesca Andriani, Giulia Bertolini, Federica Facchinetti, Erika Baldoli, Massimo Moro, Patrizia Casalini, Roberto Caserini, Ugo Pastorino, Gabriella Sozzi, Luca Roz. Conversion to stem cell state in response to microenvironmental cues is regulated by balance between epithelial and mesenchymal features in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1395. doi:10.1158/1538-7445.AM2015-1395</jats:p