Original Article Biomarkers of erlotinib response in non-small cell lung cancer tumors that do not harbor the more common epidermal growth factor receptor mutations

Abstract: Non-small cell lung cancer (NSCLC) represents approximately 85% of all lung cancers, which are the leading cause of cancer-related deaths in the world. Tyrosine kinase inhibitors such as erlotinib represent one therapeutic options presently recommended for tumors produced by activating mutations in the gene coding of epidermal growth factor receptor (EGFR). The aim of this study is the identification of possible biomarkers for tumor sensitivity to erlotinib in the absence of the main EGFR mutations. The erlotinib sensitivity of cells isolated from 41 untreated NSCLC patients was determined and compared with the presence of the more frequent EGFR mutations. Several patients had tumor cells highly sensitive to erlitinib in the absence of the EGFR mutations analyzed. The gene expression profile of 3 erlotinib-sensitive tumors was compared with that of 4 resistant tumors by DNA microarray hybridization. Sixteen genes were expressed at significantly higher levels in the resistant tumors than in the sensitive tumors. The possible correlation between erlotinib sensitivity and the expression of these genes was further analyzed using the data for the NSCLC, breast cancer and colon cancer cell lines of the NCI60 collection. The expression of these genes was correlated with the overall survival of 5 patients treated with erlotinib, according to The Cancer Genome Atlas (TCGA) database. Overlapping groups of 7, 5 and 3 genes, including UGT1A6, TRIB3, MET, MMP7, COL17A1, LCN2 and PTPRZ1, whose expression correlated with erlotinib activity was identified. In particular, low MET expression levels showed the strongest correlation

SARS-CoV-2 accessory proteins involvement in inflammatory and profibrotic processes through IL11 signaling

SARS-CoV-2, the cause of the COVID-19 pandemic, possesses eleven accessory proteins encoded in its genome. Their roles during infection are still not completely understood. In this study, transcriptomics analysis revealed that both WNT5A and IL11 were significantly up-regulated in A549 cells expressing individual accessory proteins ORF6, ORF8, ORF9b or ORF9c from SARS-CoV-2 (Wuhan-Hu-1 isolate). IL11 is a member of the IL6 family of cytokines. IL11 signaling-related genes were also differentially expressed. Bioinformatics analysis disclosed that both WNT5A and IL11 were involved in pulmonary fibrosis idiopathic disease and functional assays confirmed their association with profibrotic cell responses. Subsequently, data comparison with lung cell lines infected with SARS-CoV-2 or lung biopsies from patients with COVID-19, evidenced altered profibrotic gene expression that matched those obtained in this study. Our results show ORF6, ORF8, ORF9b and ORF9c involvement in inflammatory and profibrotic responses. Thus, these accessory proteins could be targeted by new therapies against COVID-19 disease

Metabolic and mitochondria alterations induced by SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10

Antiviral signaling, immune response and cell metabolism in human body are dysregulated by SARS-CoV-2, the causative agent of the COVID-19. Here, we show that SARS-CoV-2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 induce a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. While all four ORFs caused mitochondrial fragmentation and altered mitochondrial function, only ORF3a and ORF9c induced a marked structural alteration in mitochondrial cristae. ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes. In contrast, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes for proteins with critical mitochondrial functions and morphology. Genome-Scale Metabolic Models predicted common and private metabolic flux reprogramming, notably a depressed amino acid metabolism, and an enhanced metabolism of specific lipids distinctly induced by ORF3a. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS-CoV-2 accessory proteins that may be exploited to identify new targets for intervention.This research work was funded by the European Commission – NextGenerationEU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI+ Salud Global) (COVID-19-117 and SGL2103015), Junta de Andalucía (CV20-20089), Spanish Ministry of Science project (PID2021-123399OB-I00), the Agency for Management of University and Research Grants from Generalitat de Catalunya-AGAUR (2020PANDE00048 and 2021SGR00350) and ICREA foundation (ICREA-Academia-2021 to MC) of Generalitat de Catalunya, and an AESi grant of the Instituto de Salud Carlos III (PI20CIII-00014). TGG is recipient of a Ramón y Cajal contract funded by MCIN/AEU/10.13039/501100011033 and NextGeneration EU/PRTR.N

Estudio de la resistencia a quimioterapia en células con características de cancer stem cells (CSC) derivadas de cáncer no microcítico de pulmón

Memoria que presenta Blanca Díes López-Ayllón Licenciada en Biología, para optar al grado de Doctor por la Universidad Autónoma de Madrid (Departamento de Bioquímica) y realizada en el Instituto de Investigaciones Biomédicas Alberto Sols del CSIC.[ES]: El cáncer de pulmón es uno de los cánceres con mayor mortalidad, y en concreto el cáncer no microcítico de pulmón (CNMP) comprende más del 80% de los casos. Actualmente, los compuestos más utilizados en quimioterapia para este tipo de cáncer, son los compuestos derivados del platino, entre ellos el Cisplatino o CDDP. Sin embargo, la adquisición de resistencia a estos compuestos limita en gran medida la eficacia de los tratamientos, ya que en muchos casos no consiguen eliminar definitivamente el tumor y un porcentaje bastante alto de pacientes acaban desarrollando recidivas. Recientemente, la teoría de las células iniciadoras tumorales o cancer stem cells (CSC) nos ha permitido tomar una nueva perspectiva en el tratamiento contra el cáncer. Según esta teoría, la formación de un tumor se llevaría a cabo por la proliferación y diferenciación de una pequeña población de células “madre”, que serían resistentes a los tratamientos utilizados actualmente en clínica, y, por tanto, responsables de las recidivas. En este trabajo hemos generado células resistentes a CDDP y hemos aislado células CSC a partir de líneas celulares de CNMP, para estudiar la relación existente entre ambas poblaciones celulares. Nuestros resultados muestran que el tratamiento con una única dosis de CDDP genera células con una resistencia estable al fármaco. Por otro lado, células cultivadas en un medio selectivo y en condiciones de baja adherencia, presentan comportamientos de células iniciadoras tumorales o CSC. Además, estas células son más resistentes a CDDP y poseen un fenotipo más invasivo que las células de partida. Tanto las células CSC como las células resistentes presentan cambios de expresión de genes relacionados con invasión celular y angiogénesis, mientras que la expresión de determinados marcadores descritos previamente para las células CSC, parece estar más relacionada con la adquisición de resistencia a CDDP. Los ensayos in vivo demuestran que existe una relación entre la proporción de células CSC, la agresividad del tumor, y el aumento de la capacidad metastásica de las células tumorales. De hecho, los datos sugieren que las células CSC no son más tumorogénicas, sino que aumentan la tumorogenicidad de otras células cuando están en la proporción adecuada. Sin embargo, las células resistentes parecen tener mayor afinidad por metastatizar en hueso. Nuestros resultados concuerdan con otros estudios realizados con células CSC de CNMP aisladas por otros métodos, y sugieren que la adquisición de resistencia y el fenotipo CSC son cualidades independientes pero que están relacionadas entre sí dentro del proceso de invasión celular y metástasis. Posteriormente se ha estudiado la resistencia a CDDP y Erlotinib (Tarceva®) de células aisladas a partir de muestras quirúrgicas de pacientes con CNMP. Los resultados obtenidos indican una posible correlación entre la sensibilidad a CDDP y la presencia de células CSC, y también han permitido identificar varios posibles marcadores de resistencia a Erlotinib.[EN]: Lung cancer is one of the cancer types that produces more mortality over the World, and, in particular, non-small cell lung cancer (NSCLC) covers more than 80% of these cases. Nowadays, the compounds more frequently used for treatment of this tumor are those derived from platinum, such as cisplatin (CDDP). However, cisplatin-acquired resistance limits greatly the efficiency of this treatment, because the tumor is not completely eliminated and many patients regenerate the tumor and develop metastasis. Recently, the cancer stem cells (CSC) theory has offered a new perspective in cancer therapy. According to this theory, tumor formation takes place because of the proliferation and differentiation of a small “stem” cells population, which are therapy resistant, and so, responsibles for cancer recurrence. Our objective was to generate CDDP-resistant cells and CSC from NSCLC to study the relationship between both cell populations. Our results show that only one dose of CDDP can generate cells with stable-resistance to drug, and that cells cultured in non-adherent conditions with a selective media, have CSC behavior and are also more resistant to CDDP. Besides, these cells have more invasive and metastatic phenotype. Furthermore, both CSC and resistant cells presented gene expression changes related to cellular invasion and angiogenesis, whereas the expression of some CSC markers described previously, seems to be more connected to acquired-resistance. In vivo assays demonstrated the existence of a relationship between CSC proportion, tumor aggressiveness and metastatic capacity of tumor cells. In fact, our data show that CSC are not more tumorogenic, but they increase the tumorogenicity of other cells when they are present in a suitable proportion. However, resistant cells seem to have more affinity for bone metastasis. Our results are in agreement with studies of CSC from NSCLC isolated by different methods, and suggest that resistance acquisition and CSC phenotype are independent properties but they are connected and related to cellular invasion and metastasis. We have also studied the resistance of cells isolated from CNMP patients to CDDP and Erlotinib (Tarceva®). The results obtained indicate a possible relationship between CDDP resistance and the presence of CSC, and also allowed the identification of several possible markers of resistance to Erlotinib.Peer Reviewe

A role for cancer stem cells in drug resistance and metastasis in non-small-cell lung cancer

The cancer stem cell (CSC) theory is currently a very important fi eld in cancer research. This theory states that tumours are organised in a hierarchical manner with a subpopulation of limited number called CSCs with the ability to self-renew and undergo asymmetrical divisions, giving rise to a differentiated progeny that represents most of the tumour populations. CSCs are metastatic and chemoresistant, two features that very likely contribute to the poor response of locally advanced lung cancer. CSCs have been identifi ed in non-small-cell lung cancer cell lines as well as those from patient primary samples. A correlation has been found in terms of chemoresistance and bad prognosis in patient-derived samples enriched with CSCs, indicating that these cells are an important target for future therapy combinations. Therefore, understanding the biology and exploring cell markers and signalling pathways specifi c for CSCs of lung cancer may help in achieving progress in the treatment of the disease.Peer Reviewe

Cancer stem cells and cisplatin-resistant cells isolated from non-small-lung cancer cell lines constitute related cell populations

Lung cancer is the top cause of cancer-related deceases. One of the reasons is the development of resistance to the chemotherapy treatment. In particular, cancer stem cells (CSCs), can escape treatment and regenerate the bulk of the tumor. In this article, we describe a comparison between cancer cells resistant to cisplatin and CSCs, both derived from the non-small-cell lung cancer cell lines H460 and A549. Cisplatin-resistant cells were obtained after a single treatment with the drug. CSCs were isolated by culture in defined media, under nonadherent conditions. The isolated CSCs were clonogenic, could be differentiated into adherent cells and were less sensitive to cisplatin than the original cells. Cisplatin resistant and CSCs were able to generate primary tumors and to metastasize when injected into immunodeficient Nu/Nu mice, although they formed smaller tumors with a larger latency than untreated cells. Notably, under appropriated proportions, CSCs synergized with differentiated cells to form larger tumors. CSCs also showed increased capacity to induce angiogenesis in Nu/Nu mice. Conversely, H460 cisplatin-resistant cells showed increased tendency to develop bone metastasis. Gene expression analysis showed that several genes involved in tumor development and metastasis (EGR1, COX2, MALAT1, AKAP12, ADM) were similarly induced in CSC and cisplatin-resistant H460 cells, in agreement with a close similarity between these two cell populations. Cells with the characteristic growth properties of CSCs were also isolated from surgical samples of 18 out of 44 lung cancer patients. A significant correlation (P = 0.028) was found between the absence of CSCs and cisplatin sensitivity.B. D. López-Ayllon was supported by a Roche unrestricted grant. Supported by FIS PS09/00472, PI12/00386, PI11/00949; PI11/00537, and “Miguel Servet” financial support to Ibanez de Caceres, I (CP 08/000689; PI- 717).Peer Reviewe

Metabolic and mitochondria alterations induced by SARS‐CoV‐2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10

Publication status: PublishedFunder: ICREA foundation (ICREA‐Academia‐2021 to MC) of Generalitat de CatalunyaFunder: Serra Húnter FellowFunder: Ramón y Cajal contractAbstractAntiviral signaling, immune response and cell metabolism are dysregulated by SARS‐CoV‐2, the causative agent of COVID‐19. Here, we show that SARS‐CoV‐2 accessory proteins ORF3a, ORF9b, ORF9c and ORF10 induce a significant mitochondrial and metabolic reprogramming in A549 lung epithelial cells. While ORF9b, ORF9c and ORF10 induced largely overlapping transcriptomes, ORF3a induced a distinct transcriptome, including the downregulation of numerous genes with critical roles in mitochondrial function and morphology. On the other hand, all four ORFs altered mitochondrial dynamics and function, but only ORF3a and ORF9c induced a marked alteration in mitochondrial cristae structure. Genome‐Scale Metabolic Models identified both metabolic flux reprogramming features both shared across all accessory proteins and specific for each accessory protein. Notably, a downregulated amino acid metabolism was observed in ORF9b, ORF9c and ORF10, while an upregulated lipid metabolism was distinctly induced by ORF3a. These findings reveal metabolic dependencies and vulnerabilities prompted by SARS‐CoV‐2 accessory proteins that may be exploited to identify new targets for intervention.</jats:p

Tumor stem cells fuse with monocytes to form highly invasive tumor-hybrid cells

The ‘cancer cell fusion’ theory is controversial due to the lack of methods available to identify hybrid cells and to follow the phenomenon in patients. However, it seems to be one of the best explanations for both the origin and metastasis of primary tumors. Herein, we co-cultured lung cancer stem cells with human monocytes and analyzed the dynamics and properties of tumor-hybrid cells (THC), as well as the molecular mechanisms beneath this fusion process by several techniques: electron-microscopy, karyotyping, CRISPR-Cas9, RNA-seq, immunostaining, signaling blockage, among others. Moreover, mice models were assessed for in vivo characterization of hybrids colonization and invasiveness. Then, the presence of THCs in bloodstream and samples from primary and metastatic lesions were detected by FACS and immunofluorescence protocols, and their correlations with TNM stages established. Our data indicate that the generation of THCs depends on the expression of CD36 on tumor stem cells and the oxidative state and polarization of monocytes, the latter being strongly influenced by microenvironmental fluctuations. Highly oxidized M2-like monocytes show the strongest affinity to fuse with tumor stem cells. THCs are able to proliferate, colonize and invade organs. THC-specific cell surface signature CD36+CD14+PANK+ allows identifying them in matched primary tumor tissues and metastases as well as in bloodstream from patients with lung cancer, thus functioning as a biomarker. THCs levels in circulation correlate with TNM classification. Our results suggest that THCs are involved in both origin and spread of metastatic cells. Furthermore, they might set the bases for future therapies to avoid or eradicate lung cancer metastasis.Research in the laboratory of EL-C was supported by “Fundación para la Investigación Biomédica La Paz Hospital” ((FIBHULP) and “Fundación Familia Alonso”.Peer reviewe