Prognostic signature based on mitochondria quality control proteins for the prediction of lung adenocarcinoma patients survival.

Lung cancer is the leading cause of cancer mortality worldwide. In recent years, the incidence of lung cancer subtype lung adenocarcinoma (LUAD) has steadily increased. Mitochondria, as a pivotal site of cell bioenergetics, metabolism, cell signaling, and cell death, are often dysregulated in lung cancer cells. Mitochondria maintenance and integrity depend on mitochondrial quality control proteins (MQCPs). During lung cancer progression, the levels of MQCPs could change and promote cancer cell adaptation to the microenvironment and stresses. Here, univariate and multivariate proportional Cox regression analyses were applied to develop a signature based on the level of MQCPs (dimeric form of BNIP3, DRP1, and SIRT3) in tumorous and non-tumorous samples of 80 patients with LUAD. The MQCP signature could be used to separate the patients with LUAD into high- and low-risk groups. Survival analysis indicated that patients in the high-risk group had dramatically shorter overall survival compared with the low-risk patients. Moreover, a nomogram combining clinicopathologic features and the MQCP signature was constructed and validated to predict 1-, 3-, and 5-year overall survival of the patients. Thus, this study presents a novel signature based on MQCPs as a reliable prognostic tool to predict overall survival for patients with LUAD.

Role of macrophages in progression of colorectal cancer: a contrast with the traditional paradigm.

The phenotype of tumor-associated macrophages may be critical for tumor immunity, angiogenesis, and clinical disease outcome. Here, we elucidated the prognostic significance of the neovasculature and macrophages in colorectal cancer. We analyzed the effect of M2 macrophage density on the clinical behavior of 151 primary colorectal carcinomas using CD206 as a marker for type 2 macrophages. Triple immunohistochemical staining (ERG, SMA, and podoplanin) was used for microvessel evaluation. We found that M2 macrophages in colorectal cancer did not have a direct association with metastatic behavior. However, high numbers of CD206 tumor-associated macrophages correlated positively with recurrence-free interval duration (P=0.005). Fewer macrophages in the tumor microenvironment resulted in insufficient coverage of newly formed vessels by pericytes (P=0.011), and a high number of pericyte-impaired microvessels correlated with metastatic behavior (P<0.001). These results suggested that type 2 macrophages had a role in limiting the metastatic process by affecting vascular maturity and normalization. These findings contribute to understanding complex interactions in the tumor microenvironment and the clinical behavior of colorectal cancer.

Macrophage Phenotype in Combination with Tumor Microbiome Composition Predicts RCC Patients' Survival: A Pilot Study.

The identification of new prognostic markers of renal cell carcinoma (RCC) is an urgent problem in oncourology. To investigate the potential prognostic significance of tumor microbiome and stromal inflammatory markers, we studied a cohort of 66 patients with RCC (23 clear cell RCC, 19 papillary RCC and 24 chromophobe RCC). The microbiome was analyzed in tumor and normal tissue by 16S rRNA amplicon sequencing. Characterization of the tumor stroma was performed using immunohistochemistry. A significant difference in alpha diversity was demonstrated between normal kidney tissue and all types of RCC. Further, we demonstrated that the bacterial burden was higher in adjacent normal tissue than in a tumor. For the first time, we demonstrated a significant correlation between bacterial burden and the content of PU.1+ macrophages and CD66b+ neutrophils in kidney tumors. Tumors with high content of PU.1+ cells and CD66b+ cells in the stroma were characterized by a lower bacterial burden. In the tumors with high bacterial burden, the number of PU.1+ cells and CD66b+ was associated with a poor prognosis. The identified associations indicate the great prognostic potential of a combined tumor microbiome and stromal cell analysis.

CHID1 Is a Novel Prognostic Marker of Non-Small Cell Lung Cancer.

There is an urgent need for identification of new prognostic markers and therapeutic targets for non-small cell lung cancer (NSCLC). In this study, we evaluated immune cells markers in 100 NSCLC specimens. Immunohistochemical analysis revealed no prognostic value for the markers studied, except CD163 and CD206. At the same time, macrophage markers iNOS and CHID1 were found to be expressed in tumor cells and associated with prognosis. We showed that high iNOS expression is a marker of favorable prognosis for squamous cell lung carcinoma (SCC), and NSCLC in general. Similarly, high CHID1 expression is a marker of good prognosis in adenocarcinoma and in NSCLC in general. Analysis of prognostic significance of a high CHID1/iNOS expression combination showed favorable prognosis with 20 months overall survival of patients from the low CHID1/iNOS expression group. For the first time, we demonstrated that CHID1 can be expressed by NSCLC cells and its high expression is a marker of good prognosis for adenocarcinoma and NSCLC in general. At the same time, high expression of iNOS in tumor cells is a marker of good prognosis in SCC. When used in combination, CHID1 and iNOS show a very good prognostic capacity for NSCLC. We suggest that in the case of lung cancer, tumor-associated macrophages are likely ineffective as a therapeutic target. At the same time, macrophage markers expressed by tumor cells may be considered as targets for anti-tumor therapy or, as in the case of CHID1, as potential anti-tumor agents.

Bak and Bcl-xL Participate in Regulating Sensitivity of Solid Tumor Derived Cell Lines to Mcl-1 Inhibitors.

BH3 mimetics represent a promising tool in cancer treatment. Recently, the drugs targeting the Mcl-1 protein progressed into clinical trials, and numerous studies are focused on the investigation of their activity in various preclinical models. We investigated two BH3 mimetics to Mcl-1, A1210477 and S63845, and found their different efficacies in on-target doses, despite the fact that both agents interacted with the target. Thus, S63845 induced apoptosis more effectively through a Bak-dependent mechanism. There was an increase in the level of Bcl-xL protein in cells with acquired resistance to Mcl-1 inhibition. Cell lines sensitive to S63845 demonstrated low expression of Bcl-xL. Tumor tissues from patients with lung adenocarcinoma were characterized by decreased Bcl-xL and increased Bak levels of both mRNA and proteins. Concomitant inhibition of Bcl-xL and Mcl-1 demonstrated dramatic cytotoxicity in six of seven studied cell lines. We proposed that co-targeting Bcl-xL and Mcl-1 might lead to a release of Bak, which cannot be neutralized by other anti-apoptotic proteins. Surprisingly, in Bak-knockout cells, inhibition of Mcl-1 and Bcl-xL still resulted in pronounced cell death, arguing against a sole role of Bak in the studied phenomenon. We demonstrate that Bak and Bcl-xL are co-factors for, respectively, sensitivity and resistance to Mcl-1 inhibition.

Immunosuppressive Phenotype of Esophagus Tumors Stroma.

BACKGROUND: Tumor-associated macrophages (TAMs) and tumor-infiltrating lymphocytes (TILs) contribute significantly to the development of immunosuppressive properties of a tumor. In this study, we performed immunohistochemical analysis of immune cells of esophageal tumors stroma. METHODS: Paraffin-embedded tissue specimens from 48 esophageal squamous cell carcinoma (ESCC) patients were retrospectively collected for immunohistochemical analysis of stromal cells. For staining of macrophages, CD68, CD163, CD206, PU.1, and iNOS were used. For T cell detection, CD8, CD3, and FOXP3 were used. Also, we performed staining for PD-L1 that can be expressed on TAMs and tumor cells. Clinicopathological and survival data were collected and analyzed using the χ 2 and Fisher exact tests, Kaplan-Meier curves, and the log-rank test. The correlation analysis was performed with Spearman's rank correlation coefficient. RESULTS: We found that FOXP3 expression was associated with age (p = 0.042) and iNOS expression was associated with the disease stage (p = 0.044). In addition, FOXP3 and CD163 appeared to be markers of good prognosis (HR = 0.4420, p = 0.0325, and HR = 0.4447, p = 0.0456, respectively). Significant association between PU.1+ and CD68+ macrophages (r = 0.833; p ≤ 0.001) and between PU.1+ and CD163+ macrophages (r = 0.500; p ≤ 0.001) was established; positive association between PU.1 and CD206 expression was also observed (r = 0.250; p = 0.043). CONCLUSIONS: Large amounts of CD163+ macrophages and FOXP3+ Т cells appear to be markers of good prognosis of ESCC. The number of PU.1+ macrophages strongly correlates with the number of CD68+ macrophages; therefore, usage of PU.1 as a potential macrophage marker can be recommended for esophageal tumors.

Pyrazoles: 'one-pot' synthesis from arenes and carboxylic acids.

A rapid and efficient method for 'one-pot' synthesis of pyrazoles from (hetero)arenes and carboxylic acids via successive formation of ketones and ß-diketones followed by heterocyclization with hydrazine has been developed. The utility of the RCOOH/TfOH/TFAA acylation system for intermediate production of ketones and 1,3-diketones is a key feature of this approach. The preliminary evaluation of the anticancer activity of the synthesized pyrazoles is performed.

Human Lung Microbiome on the Way to Cancer.

Recent research on cancer-associated microbial communities led to the accumulation of data on the interplay between bacteria, immune and tumor cells, the pathways of bacterial induction of carcinogenesis, and its meaningfulness for medicine. Microbial communities that have any kind of impact on tumor progression and microorganisms associated with tumors have been defined as oncobiome. Over the last decades, a number of studies were dedicated to Helicobacter pylori and its role in the progression of stomach tumors, so this correlation can be regarded as proven. Involvement of bacteria in the induction of lung cancer has been largely ignored for a long time, though some correlations between this type of cancer and lung microbiome were established. Despite the fact that in the present the microbial impact on lung cancer progression has many confirmations, the underlying mechanisms are poorly understood. Microorganisms can contribute to tumor initiation and progression through production of bacteriotoxins and other proinflammatory factors. The purpose of this review is to organize the available data on lung cancer microbiome and its role in malignant tumor progression.

Development and Characterization of a Novel Monoclonal Antibody Against Chitinase-like Protein CHID1 Applicable for Immunohistochemistry on Formalin Fixed Paraffin-Embedded Sections.

CHID1 has been recently described as a predictive marker of different malignant tumors. Thus, monoclonal antibodies (mAbs) for CHID1 detection in different human liquids and in tissues are an important tool for the diagnosis of CHID1-positive cancers. However, only few mAbs have been established to date. In this study we describe the generation of a new hybridoma clone 3D4 producing anti-CHID1 antibodies. 3D4 mAb specifically binds human CHID1 and was successfully used in enzyme-linked immunosorbent assay, immunoblotting, immunofluorescence on paraformaldehyde-fixed cells, and in immunohistochemistry of paraffin-embedded tissue specimens. These results indicate that this new anti-CHID1 mAb 3D4 will be useful in the diagnosis of CHID1-related cancers and is a strong tool for both basic and clinical research on chitinase-like proteins.

A Novel Monoclonal Antibody Against Alpha-Methylacyl-CoA Racemase Applicable for Paraffin-Embedded Tissues and Diagnostics of Prostate Cancer.

AMACR (alpha-methylacyl-CoA racemase) has been recently described as a prostate cancer-specific gene that encodes a protein involved in the beta-oxidation of branched chain fatty acids. Expression of AMACR protein is found in prostatic adenocarcinoma, but not in benign prostatic tissue. Thus, monoclonal antibodies (mAbs) for AMACR detection are an important tool for the diagnosis of AMACR-positive cancers. However, only a few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we describe the generation of a new hybridoma clone G8 producing anti-AMACR antibodies. G8 mAb specifically binds human AMACR and was successfully used in immunoblotting and immunofluorescence on paraformaldehyde-fixed cells and in IHC of paraffin-embedded tumor specimens. These results indicate that this new anti-AMACR mAb G8 would be useful in the diagnosis of AMACR-related cancers and would be a strong tool in both basic and clinical research on AMACR.

Tumor Associated Macrophages in Kidney Cancer.

Tumor associated macrophages (TAMs) are an important element of tumor stroma. They originate from blood monocytes attracted by chemokines and cytokines produced by tumor cells and, being instructed by tumor microenvironment, develop into potent tumor-supporting cell population. TAMs were demonstrated to directly stimulate tumor cell proliferation and to promote angiogenesis. Further TAMs provide for efficient immune escape by producing immunosuppressive cytokines and facilitate tumor dissemination by producing extracellular matrix remodeling enzymes. In renal cell carcinoma (RCC), numerous studies were performed for elucidation of the role of TAM in tumor progression. Using pan-macrophages marker CD68 and type 2 macrophage (M2) markers CD163 and CD206, it was demonstrated that increased density of TAMs is associated with poor survival of patients. Although most of the studies are focused on M2 population in RCC, several markers rather typical for type 1 macrophages (M1) were also characterized. Macrophages isolated from RCC tumors were shown to produce proinflammatory cytokines TNFα, IL-1ß, IL-6, and CCL2. It can be concluded that RCC is an excellent example of a tumor with hybrid phenotype of TAMs that share both M1 and M2 properties. Moreover, TAMs seem to be an attractive therapeutic target as well. Further investigations are needed for identification of RCC-specific TAM markers with high predictive capacity and/or suitable for therapeutic targeting.

Arf6 promotes cell proliferation via the PLD-mTORC1 and p38MAPK pathways.

The small G-protein ADP-ribosylation factor 6 (Arf6) belongs to the Ras GTPases superfamily and is mostly known for its actin remodeling functions and involvement in the processes of plasma membrane reorganization and vesicular transport. The majority of data indicates that Arf6 contributes to cancer progression through activation of cell motility and invasion. Alternatively, we found that the expression of a wild-type or a constitutively active Arf6 does not influence tumor cell motility and invasion but instead significantly stimulates cell proliferation and activates phospholipase D (PLD). Conversely the expression of a mutant Arf6 (Arf6N48I), that is, unable to interact with PLD has no effect on proliferation but promotes motility, invasion, and matrix degradation by uPA extracellular proteinase. Studying the mechanisms of Arf6-dependent stimulation of cell proliferation, we found some signaling pathways contributing to Arf6 promitogenic activity. Namely, we showed that Arf6 in a PLD-mTORC1-dependent manner activates S6K1 kinase, a well-known regulator of mitogen-stimulated translation initiation. Furthermore, we demonstrated an Arf6-dependent phosphorylation of mTORC1 downstream targets, 4E-BP1 and ribosomal S6 protein, confirming an existence of Arf6-PLD-mTORC1-S6K1/4E-BP1 signaling pathway and also demonstrated its impact on proliferation stimulation. Next, we found that Arf6 activation potentiates Erk1/2 and p38MAP kinases phosphorylation. Surprisingly, p38 opposite to Erk1/2 significantly contributes to Arf6-dependent proliferation increase promoting S6 ribosomal protein phosphorylation at Ser235/236 residues. Therefore, we demonstrated Arf6 proliferation stimulating activity and revealed PLD-mTORC1 and p38MAP kinase as Arf6 partners mediating promitogenic activity. These results highlight a new aspect of Arf6 functioning in cancer cell biology.