Tumor Tissues Of Cancer Patients Research Articles

Shugoshin 1 expression in various cancers: a potential target for therapy.

Shugoshin 1 (SGO1) is one of the Shugoshin (guardian spirit) family proteins, which is reported to be majorly involved in the protection of centromeres and proper segregation of chromosomes during cell division. Recent studies found that the altered expression of SGO1 is associated with various cancers and genetic disorders, and suggested as a target for therapy. In the present study, we have reviewed the available literature on SGO1 gene and protein expression in various cancer-cell lines, animal models and cancer patients, and targeting SGO1 with siRNA/shRNA. A significant increase in the expression of SGO1 mRNA and protein levels were observed in prostate, renal, lung, breast, neuroblastoma, leukemia, hepatocellular, and colon cancer-cell lines and the levels were associated with increased cellular proliferation, invasion, and metastasis. The altered SGO1 levels were observed in SGO1 knockout/haploinsufficient mice compared to wild type and the levels were associated with increased chromosome instability and tumorigenesis. Consistent with cell lines, higher SGO1 expression was also observed in tumor tissues of cancer patients compared to adjacent normal tissue and the levels were positively correlated with tumor stage, grade, size, and hormonal status. Higher SGO1 expression was related to resistance to chemotherapeutic agents and the knockdown of SGO1 increased sensitivity to those agents. Furthermore, targeting SGO1 with siRNA/shRNA reduced the expression of SGO1 and proliferation, and induced apoptosis of cancer cells. Overall, the SGO1 expression levels were significantly higher in various cancers, and targeting SGO1 with siRNA and shRNA reduced the levels of SGO1, proliferation and metastasis of cancers.

Th17 cells target the metabolic miR-142-5p-succinate dehydrogenase subunit C/D (SDHC/SDHD) axis, promoting invasiveness and progression of cervical cancers.

During cervical carcinogenesis, T-helper (Th)-17 cells accumulate in the peripheral blood and tumor tissues of cancer patients. We previously demonstrated that Th17 cells are associated with therapy resistance as well as cervical cancer metastases and relapse; however, the underlying Th17-driven mechanisms are not fully understood. Here, using microarrays, we found that Th17 cells induced an epithelial-to-mesenchymal transition (EMT) phenotype of cervical cancer cells and promoted migration and invasion of 2D cultures and 3D spheroids via induction of microRNA miR-142-5p. As the responsible mechanism, we identified the subunits C and D of the succinate dehydrogenase (SDH) complex as new targets of miR-142-5p and provided evidence that Th17-miR-142-5p-dependent reduced expression of SDHC and SDHD mediated enhanced migration and invasion of cancer cells using small interfering RNAs (siRNAs) for SDHC and SDHD, and miR-142-5p inhibitors. Consistently, patients exhibited high levels of succinate in their serum associated with lymph node metastases and diminished expression of SDHD in patient biopsies correlated with increased numbers of Th17 cells. Correspondingly, a combination of weak or negative SDHD expression and a ratio of Th17/CD4+ T cells > 43.90% in situ was associated with reduced recurrence-free survival. In summary, we unraveled a previously unknown molecular mechanism by which Th17 cells promote cervical cancer progression and suggest evaluation of Th17 cells as a potential target for immunotherapy in cervical cancer.

Doxorubicin resistance in breast cancer is mediated via the activation of FABP5/PPARγ and CaMKII signaling pathway.

Breast cancer is the most prevalent malignancy among women. Doxorubicin (Dox) resistance was one of the major obstacles to improving the clinical outcome of breast cancer patients. The purpose of this study was to investigate the relationship between the FABP signaling pathway and Dox resistance in breast cancer. The resistance property of MCF-7/ADR cells was evaluated employing CCK-8, Western blot (WB), and confocal microscopy techniques. The glycolipid metabolic properties of MCF-7 and MCF-7/ADR cells were identified using transmission electron microscopy, PAS, and Oil Red O staining. FABP5 and CaMKII expression levels were assessed through GEO and WB approaches. The intracellular calcium level was determined by flow cytometry. Clinical breast cancer patient's tumor tissues were evaluated by immunohistochemistry to determine FABP5 and p-CaMKII protein expression. In the presence or absence of FABP5 siRNA or the FABP5-specific inhibitor SBFI-26, Dox resistance was investigated utilizing CCK-8, WB, and colony formation methods, and intracellular calcium level was examined. The binding ability of Dox was explored by molecular docking analysis. The results indicated that the MCF-7/ADR cells we employed were Dox-resistant MCF-7 cells. FABP5 expression was considerably elevated in MCF-7/ADR cells compared to parent MCF-7 cells. FABP5 and p-CaMKII expression were increased in resistant patients than in sensitive individuals. Inhibition of the protein expression of FABP5 by siRNA or inhibitor increased Dox sensitivity in MCF-7/ADR cells and lowered intracellular calcium, PPARγ, and autophagy. Molecular docking results showed that FABP5 binds more powerfully to Dox than the known drug resistance-associated protein P-GP. In summary, the PPARγ and CaMKII axis mediated by FABP5 plays a crucial role in breast cancer chemoresistance. FABP5 is a potentially targetable protein and therapeutic biomarker for the treatment of Dox resistance in breast cancer.

Chemoradiotherapy-induced increase in Th17 cell frequency in cervical cancer patients is associated with therapy resistance and early relapse.

Cervical cancer therapy is still a major clinical challenge, as patients substantially differ in their response to standard treatments, including chemoradiotherapy (CRT). During cervical carcinogenesis, T‐helper (Th)‐17 cells accumulate in the peripheral blood and tumor tissues of cancer patients and are associated with poor prognosis. In this prospective study, we find increased Th17 frequencies in the blood of patients after chemoradiotherapy and a post‐therapeutic ratio of Th17/CD4+ T cells > 8% was associated with early recurrence. Furthermore, Th17 cells promote resistance of cervical cancer cells toward CRT, which was dependent on the AKT signaling pathway. Consistently, patients with high Th17 frequencies in pretherapeutic biopsies exhibit lower response to primary CRT. This work reveals a key role of Th17 cells in CRT resistance and elevated Th17 frequencies in the blood after CRT correspond with early recurrence. Our results may help to explain individual treatment responses of cervical cancer patients and suggest evaluation of Th17 cells as a novel predictive biomarker for chemoradiotherapy responses and as a potential target for immunotherapy in cervical cancer.

A COMPARATIVE STUDY ASSESSING THE DIFFERENT PATTERNS OF RADIATION REACTIONS IN HEAD AND NECKCANCER PATIENTS UNDERGOING RADIATION THERAPY AT DIFFERENT TIMES OF THE DAY-IN THE MORNING VERSUS EVENING

Background: Human oral mucosa, is a prime target for radiation induced toxicity in patients undergoing radiotherapy for head and neck cancer. The oral cavity is highly susceptible to direct and indirect toxic effects of cancer chemotherapy and ionizing radiation. In our study we investigated the role of chronotherapy with respect to radiation induced mucositis occurring in the normal oral mucosa and treatment response in tumor tissue in cancer patients undergoing radiation therapy to head and neck region. Materials and Methods: This non-randomized clinical study was done to compare the two different timing schedules in the management of head and neck cancer by external beam radiation therapy by cobalt-60 (Theratron780C). In morning arm (arm A), Patients received external beam radiotherapy (EBRT) by cobalt-60 with SSD of 80cm with daily fractions, five days a week, between 8AM–10 AM whereas in evening arm (arm B), patients received EBRT by cobalt-60 with daily fractions, five days a week, between 3PM–5 PM. Both the arms received same radiation regimen. Results: The study concluded that in the morning arm the onset of oral mucositis was observed to be significantly delayed and the duration of mucositis was also significantly shorter as compared to the evening arm. Statistically significant difference in severity of mucositis was found between the two arms. Conclusion: Since there is no active measure to minimize normal tissue toxicity, chronomodulated radiotherapy would be a feasible and cost effective treatment strategy that can be put into clinical practice.

Assessing the Concordance of Genomic Alterations between Circulating-Free DNA and Tumour Tissue in Cancer Patients.

Somatic alterations to the genomes of solid tumours, which in some cases represent actionable drivers, provide diagnostic and prognostic insight into these complex diseases. Spatial and longitudinal tracking of somatic genomic alterations (SGAs) in patient tumours has emerged as a new avenue of investigation, not only as a disease monitoring strategy, but also to improve our understanding of heterogeneity and clonal evolution from diagnosis through disease progression. Furthermore, analysis of circulating-free DNA (cfDNA) in the so-called “liquid biopsy” has emerged as a non-invasive method to identify genomic information to inform targeted therapy and may also capture the heterogeneity of the primary and metastatic tumours. Considering the potential of cfDNA analysis as a translational laboratory tool in clinical practice, establishing the extent to which cfDNA represents the SGAs of tumours, particularly actionable driver alterations, becomes a matter of importance, warranting standardisation of methods and practices. Here, we assess the utilisation of cfDNA for molecular profiling of SGAs in tumour tissue across a broad range of solid tumours. Moreover, we examine the underlying factors contributing to discordance of detected SGAs between cfDNA and tumour tissue.

Genomic Profiles and Subset Characterization of CD8+terminally Differentiated Effector Memory (TEMRA) Cells from Cancer Patients

Introduction Terminally differentiated effector memory (TEMRA) cells have been recent revealed as sub-population of CD8 T cells, and they have been expected to act like memory-like properties in peripheral blood. However, the precise characteristics or function of these cells have not been defined. Particularly, these cells have been found even in cancer tissues including tumor-infiltrating effector CD8+ T cells (TILEM). The study investigated the phenotypic or genomic difference of CD8+ TEMRA cells which extracted from not only peripheral blood of healthy and cancer patients but also tumor tissues of cancer patients. Material and methods Peripheral blood or tumor tissues from normal donors and patients with lung cancer or Hodgkin lymphoma were collected. Three different sources of CD8+ TEMRA cells, which from healthy peripheral blood (hTEMRA), patient peripheral blood (pTEMRA) and TIL (TIL TEMRA), were obtained and then analyzed for phenotypic and genomic differences. CD8+ T cells were isolated using magnetic-activated cell sorting (MACS), and sub-type cells were isolated using FACS (fluorescence-activated cell sorting) based on phenotypic factors (TEMRA; CD8+ CCR7- CD45RA+). Tumor tissues from patients with lung cancer were digested with enzyme and preceded to the same manner as peripheral blood. Next-generation sequencing (NGS) was used to characterize the TEMRA by using Hiseq2000 (Illumina) on cDNA synthesized from RNA extracted from sorted cells (TEM; CD8+ CCR7- CD45RA-, TEMRA; CD8+ CCR7- CD45RA+). All experiments were performed after obtaining the informed consent approved by the institutional ethical committee (IRB). Results CD8+ TEMRA exist with similar proportion in cancer tissues as well as peripheral blood in lung cancer patients. TIL TEMRA was distinguished from regulatory T cells without expression of CD25 and foxp3. All healthy or patient TEMRA cells including TIL TEMRA showed similar expression level of exhaustion phenotypes (CTLA4, PD-1 and Tim3) (p>0.05). However, TIL TEMRA cells exhibited significantly less amounts of T cell exhaustion markers (PD-1 and Tim3) than those of TILEM cells. Genomic background of blood-derived TEMRA differs from tumor tissue-derived TEMRA based on NGS assessment. Moreover, unlike general CD8+ TILEM, which consist mostly of exhausted effector cells due to tumor-derived immunosuppression, CD8+ TIL TEMRA showed the different genomic background. The transcriptional factors (T-bet and Eomes) or genomic patterns of CD8+ TIL TEMRA represented less exhausted, proliferative and cytotoxic than CD8+ TILEM. However, CD8+ TIL TEMRA might be induced into secondary effector cells after TCR re-stimulation. Conclusion Genomic and functional characteristics of CD8+ TIL TEMRA distinct from not only TIL TEM but also peripheral TEMRA.in cancer patients. This research could define the different subset of CD8+ TILs in cancer and give a clue for different response or modulating therapeutic response of immune checkpoint inhibitor by using CD8+ TIL TEMRA cells. Figure Disclosures No relevant conflicts of interest to declare.

A flow-proteometric platform for analyzing protein concentration (FAP): Proof of concept for quantification of PD-L1 protein in cells and tissues

Protein expression level is critically related to the cell physiological function. However, current methodologies such as Western blot (WB) and Immunohistochemistry (IHC) in analyzing the protein level are rather semi-quantitative and without the information of actual protein concentration. We have developed a microfluidic technique termed a “flow-proteometric platform for analyzing protein concentration (FAP)” that can measure the concentration of a target protein in cells or tissues without the requirement of a calibration standard, e.g., the purified target molecules. To validate our method, we tested a number of control samples with known target protein concentrations and showed that the FAP measurement resulted in concentrations that well matched the actual concentrations in the control samples (coefficient of determination [R2], 0.998), demonstrating a dynamic range of concentrations from 0.13 to 130 pM of a detection for 2 min. We successfully determined a biomarker protein (for predicting the treatment response of cancer immune check-point therapy) PD-L1 concentration in cancer cell lines (HeLa PD-L1 and MDA-MB-231) and breast cancer patient tumor tissues without any prior process of sample purification and standard line construction. Therefore, FAP is a simple, faster, and reliable method to measure the protein concentration in cells and tissues, which can support the conventional methods such as WB and IHC to determine the actual protein level.

Antitumor activity of kinetochore-associated protein 2 siRNA against lung cancer patient-derived tumor xenografts.

It has been widely reported that patient-derived tumor xenografts (PDXs) are more similar to tumor tissues than conventional cancer cell lines. Kinetochore-associated protein 2 (KNTC2) is known to be upregulated specifically in tumor tissues of cancer patients and is recognized as a potential target for cancer therapy. Previously, in vivo antitumor activities of KNTC2 short interfering RNA encapsulated into a lipid nanoparticle (KNTC2-LNP) were reported in orthotopic hepatocellular carcinoma mouse models. However, it remains unclear whether KNTC2-LNP exhibits antitumor activities against lung cancer PDXs. In the present study, the antitumor activities of KNTC2-LNP were clarified in a three-dimensional culture system and a subcutaneous tumor model of lung cancer PDX, LC-60, which was resistant to erlotinib. Growth inhibitory activities of KNTC2-LNP were associated with knockdown activities. Furthermore, KNTC2-LNP also exhibited in vivo antitumor activity against another lung cancer PDX, LC-45, which was sensitive to erlotinib. These results suggest that KNTC2 is a promising target for patients with lung cancer.

Ethical frameworks for obtaining informed consent in tumour profiling: an evidence-based case for Singapore

BackgroundGenomic profiling of malignant tumours has assisted clinicians in providing targeted therapies for many serious cancer-related illnesses. Although the characterisation of somatic mutations is the primary aim of tumour profiling for treatment, germline mutations may also be detected given the heterogenous origin of mutations observed in tumours. Guidance documents address the return of germline findings that have health implications for patients and their genetic relations. However, the implications of discovering a potential but unconfirmed germline finding from tumour profiling are yet to be fully explored. Moreover, as tumour profiling is increasingly applied in oncology, robust ethical frameworks are required to encourage large-scale data sharing and data aggregation linking molecular data to clinical outcomes, to further understand the role of genetics in oncogenesis and to develop improved cancer therapies.ResultsThis paper reports on the results of empirical research that is broadly aimed at developing an ethical framework for obtaining informed consent to return results from tumour profiling tests and to share the biomolecular data sourced from tumour tissues of cancer patients. Specifically, qualitative data were gathered from 36 semi-structured interviews with cancer patients and oncology clinicians at a cancer treatment centre in Singapore. The interview data indicated that patients had a limited comprehension of cancer genetics and implications of tumour testing. Furthermore, oncology clinicians stated that they lacked the time to provide in depth explanations of the tumour profile tests. However, it was accepted from both patients and oncologist that the return potential germline variants and the sharing of de-identified tumour profiling data nationally and internationally should be discussed and provided as an option during the consent process.ConclusionsFindings provide support for the return of tumour profiling results provided that they are accompanied with an adequate explanation from qualified personnel. They also support the use of broad consent regiments within an ethical framework that promotes trust and benefit sharing with stakeholders and provides accountability and transparency in the storage and sharing of biomolecular data for research.

Abstract 826: Nrf2 confers radioresistance by regulating cancer stem trait in lung cancer cells

Abstract Lung cancer is the leading cause of cancer-related death accounting for more than 1.3 million deaths worldwide annually. Despite advances in treatment, the five-year survival rate is 2% if the cancer has metastasized to other organs. The major cause of poor prognosis of advanced lung cancer is multi-resistance to chemo/radio therapy. Therefore, it is crucial to understand the underlying mechanisms of chemo/radio resistance and develop therapeutic strategies to overcome the resistance. Transcription factor Nrf2 is a key regulator of cytoprotective genes which facilitate cellular defense against harmful insults. We found that Nrf2 and its downstream target NQO1 are highly upregulated in lung cancer patient tumor tissues as well as in lung cancer cell lines compared to adjacent normal tissues or normal cell lines. We hypothesized that the upregulated Nrf2 may be responsible for radio/chemo resistance and malignancy in lung cancer. RNA interference studies showed that knockdown of Nrf2 substantially increased the sensitivity of human lung cancer H460 and A549 cells to radiation-induced cell death. Knockdown of Nrf2 also decreased tumor sphere formation, suggesting its role in cancer stem cell (CSC) regulation which may be responsible for the radioresistance. Analyses of CSC markers including ALDH and Oct4 in control and Nrf2-knockdown H460 and A549 cells support this finding. Together, our results indicate an essential role of Nrf2 in the control and regulation of CSCs and radioresistance. Since CSCs are widely believed to be responsible for cancer chemoresistance and relapse, our novel finding on the role of Nrf2 in radiation resistance may aid in the design of novel therapeutic strategies for resistant cancers. Citation Format: Xiaoqing He, Alex Chi, Liying Wang, Yon Rojanasakul. Nrf2 confers radioresistance by regulating cancer stem trait in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 826. doi:10.1158/1538-7445.AM2017-826

Identification of miR-601 as a novel regulator in the development of pancreatic cancer

Pancreatic cancer (PC) is one of the most aggressive malignancies, with a high mortality. Distant metastasis and recurrence are the main causes of PC-related deaths. MicroRNAs (miRNAs) have been reported in the serum or tumor tissue of cancer patients, including PC, which makes them potential biomarkers. The dysfunction of many miRNAs has been linked to PC occurrence and metastasis. In the current study, we found that miR-601 expression was significantly lower in PC samples, especially in metastatic compared to non-metastatic PC tissues. Gain-of-function and loss-of-function analysis showed that miR-601 suppressed PC cell proliferation and migration. One potential mechanism is that miR-601 inhibited the expression of Sirtuin 1 (SIRT1), which is a well-known regulator of PC development. We found that overexpression of SIRT1 could reverse the effect of miR-601 on PC cells. Our investigation therefore suggests that both miR-601 and SIRT1 are possible biomarkers for the early detection, and targets for the treatment of PC.

Neutrophils in cancer development and progression: Roles, mechanisms, and implications (Review).

Neutrophils are predominant immune cells that protect the host from microbial infection. The roles of neutrophils in tumor have long been ignored due to their short life span and terminal differentiation phenotype. In recent years, emerging evidence indicates that neutrophils have phenotypic and functional plasticity. Neutrophils eliminate malignant cells by releasing the antimicrobial and cytotoxic contents in their granules or secreting immune mediators to recruit and activate other antitumor effector cells. On the contrary, tumor derived factors can convert neutrophils into a pro-tumor phenotype. Neutrophils have been shown to facilitate tumorigenesis, promote tumor growth and metastasis, stimulate tumor angiogenesis, and mediate immunosuppression. The number of neutrophils in blood and tumor tissues of cancer patients is associated with disease progression and patient outcome. In this review, we summarize the recent progress of neutrophils in the pathogenesis of cancer with an emphasis on neutrophil polarization. Better understanding of the mechanisms that regulate the dichotomy of neutrophils will not only shed light on their roles in cancer but also provide new approaches for cancer diagnosis and treatment.

Regulatory T Cells in Cancer Patients and Their Roles in Cancer Development/Progression

The immune system can protect body against malignant cell formation and cancer development. However, in some cases malignant cells survive and a tumor develops leading to cancer. The immunosuppressive function of some subsets of immune cells is involved in evading the tumor cells from immunosurveillance.In the past decade, elevated levels of regulatory T cells (Tregs) were reported in the peripheral blood and tumor tissue of cancer patients. In most cancer patients, increased levels of Tregs were correlated with poor prognosis. In contrast, increased frequencies of Tregs were associated with favorable prognosis in some cancer patients. So far, the precise roles of Tregs in carcinogenesis and cancer progression have not been addressed. Better understanding of the immunobiology of Tregs is beneficial for elucidation of their roles in cancers and innovation of more effective cancer therapy modalities. In this review, we present extensive information about the immunophenotype and functions of Tregs, mechanisms that are implicated in the recruitment, differentiation, and/or expansion ofthese cells at tumor sites, as well as their frequencies and roles in cancer patients. We also assessed their predictive value in cancer patients.

Nucleoside-catabolizing Enzymes in Mycoplasma-infected Tumor Cell Cultures Compromise the Cytostatic Activity of the Anticancer Drug Gemcitabine

The intracellular metabolism and cytostatic activity of the anticancer drug gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdC) was severely compromised in Mycoplasma hyorhinis-infected tumor cell cultures. Pronounced deamination of dFdC to its less cytostatic metabolite 2',2'-difluoro-2'-deoxyuridine was observed, both in cell extracts and spent culture medium (i.e. tumor cell-free but mycoplasma-containing) of mycoplasma-infected tumor cells. This indicates that the decreased antiproliferative activity of dFdC in such cells is attributed to a mycoplasma cytidine deaminase causing rapid drug catabolism. Indeed, the cytostatic activity of gemcitabine could be restored by the co-administration of tetrahydrouridine (a potent cytidine deaminase inhibitor). Additionally, mycoplasma-derived pyrimidine nucleoside phosphorylase (PyNP) activity indirectly potentiated deamination of dFdC: the natural pyrimidine nucleosides uridine, 2'-deoxyuridine and thymidine inhibited mycoplasma-associated dFdC deamination but were efficiently catabolized (removed) by mycoplasma PyNP. The markedly lower anabolism and related cytostatic activity of dFdC in mycoplasma-infected tumor cells was therefore also (partially) restored by a specific TP/PyNP inhibitor (TPI), or by exogenous thymidine. Consequently, no effect on the cytostatic activity of dFdC was observed in tumor cell cultures infected with a PyNP-deficient Mycoplasma pneumoniae strain. Because it has been reported that some commensal mycoplasma species (including M. hyorhinis) preferentially colonize tumor tissue in cancer patients, our findings suggest that the presence of mycoplasmas in the tumor microenvironment could be a limiting factor for the anticancer efficiency of dFdC-based chemotherapy. Accordingly, a significantly decreased antitumor effect of dFdC was observed in mice bearing M. hyorhinis-infected murine mammary FM3A tumors compared with uninfected tumors.

The Pyrimidine Nucleoside Phosphorylase of Mycoplasma hyorhinis and How It May Affect Nucleoside-Based Therapy

Mycoplasmas are opportunistic parasites and some species are suggested to preferentially colonize tumor tissue in cancer patients. We could demonstrate that the annotated thymidine phosphorylase (TP) gene in the genome of Mycoplasma hyorhinis encodes a pyrimidine nucleoside phosphorylase (PyNPHyor) that not only efficiently catalyzes thymidine but also uridine phosphorolysis. The kinetic characteristics of PyNPHyor-catalyzed nucleoside and nucleoside analogue (NA) phosphorolysis were determined. We demonstrated that the expression of such an enzyme in mycoplasma-infected cell cultures dramatically alters the activity of various anticancer/antiviral NAs such as 5-halogenated pyrimidine nucleosides, including 5-trifluorothymidine (TFT). Due to their close association with human cancers, the presence of mycoplasmas may markedly influence the therapeutic efficiency of nucleoside-based drugs.

Mycoplasma hyorhinis–Encoded Purine Nucleoside Phosphorylase: Kinetic Properties and Its Effect on the Cytostatic Potential of Purine-Based Anticancer Drugs

A mycoplasma-encoded purine nucleoside phosphorylase (designated PNPHyor) has been cloned and characterized for the first time. Efficient phosphorolysis of natural 6-oxopurine and 6-aminopurine nucleosides was observed, with adenosine the preferred natural substrate (Km = 61 µM). Several cytostatic purine nucleoside analogs proved to be susceptible to PNPHyor-mediated phosphorolysis, and a markedly decreased or increased cytostatic activity was observed in Mycoplasma hyorhinis-infected human breast carcinoma MCF-7 cell cultures (MCF-7.Hyor), depending on the properties of the released purine base. We demonstrated an ∼10-fold loss of cytostatic activity of cladribine in MCF-7.Hyor cells and observed a rapid and complete phosphorolysis of this drug when it was exposed to the supernatant of mycoplasma-infected cells. This conversion (inactivation) could be prevented by a specific PNP inhibitor. These findings correlated well with the high efficiency of PNPHyor-catalyzed phosphorolysis of cladribine to its less toxic base 2-chloroadenine (Km = 80 µM). In contrast, the cytostatic activity of nucleoside analogs carrying a highly toxic purine base and being a substrate for PNPHyor, but not human PNP, was substantially increased in MCF-7.Hyor cells (∼130-fold for fludarabine and ∼45-fold for 6-methylpurine-2'-deoxyriboside). Elimination of the mycoplasma from the tumor cell cultures or selective inhibition of PNPHyor by a PNP inhibitor restored the cytostatic activity of the purine-based nucleoside drugs. Since several studies suggest a high and preferential colonization or association of tumor tissue in cancer patients with different prokaryotes (including mycoplasmas), the data presented here may be of relevance for the optimization of purine nucleoside-based anticancer drug treatment.

Phenotypic and functional characteristics of CD4+CD39+ FOXP3+ and CD4+CD39+FOXP3neg T‐cell subsets in cancer patients

Human CD4(+) CD39(+) regulatory T (Treg) cells hydrolyze exogenous adenosine triphosphate (ATP) and participate in immunosuppressive adenosine production. They contain two T-cell subsets whose role in mediating suppression is not understood. Frequencies of both CD4(+) CD39(+) subsets were evaluated in peripheral blood lymphocytes of 57 cancer patients and in tumor infiltrating lymphocytes (TILs) of 6 patients. CD4(+) CD39(+) and CD4(+) CD39(neg) T cells isolated using immunobeads and cell sorting were cultured under various conditions. Their conversion into CD39(+) FOXP3(+) CD25(+) or CD39(+) FOX(neg) CD25(neg) cells was monitored by multiparameter flow cytometry. Hydrolysis of exogenous ATP was measured in luminescence assays. Two CD4(+) CD39(+) cell subsets differing in expression of CD25, FOXP3, CTLA-4, CD121a, PD-1, latency associated peptide (LAP), glycoprotein A repetitions predominant (GARP), and the cytokine profile accumulated with equal frequencies in the blood and tumor tissues of cancer patients. The frequency of both subsets was significantly increased in cancer. CD39 expression levels correlated with the subsets' ability to hydrolyze ATP. Conventional CD4(+) CD39(neg) T cells incubated with IL-2 + TGF-β expanded to generate CD4(+) CD39(+) FOXP3(+) Treg cells, while CD4(+) CD39(+) FOXP3(neg) CD25(neg) subset cells stimulated via the TCR and IL-2 converted to FOXP3(+) CTLA4(+) CD25(+) TGF-β-expressing Treg cells. Among CD4(+) CD39(+) Treg cells, the CD4(+) CD39(+) FOXP3(neg) CD25(neg) subset serves as a reservoir of cells able to convert to Treg cells upon activation by environmental signals.

Characterization of pyrimidine nucleoside phosphorylase of Mycoplasma hyorhinis: implications for the clinical efficacy of nucleoside analogues

In the present paper we demonstrate that the cytostatic and antiviral activity of pyrimidine nucleoside analogues is markedly decreased by a Mycoplasma hyorhinis infection and show that the phosphorolytic activity of the mycoplasmas is responsible for this. Since mycoplasmas are (i) an important cause of secondary infections in immunocompromised (e.g. HIV infected) patients and (ii) known to preferentially colonize tumour tissue in cancer patients, catabolic mycoplasma enzymes may compromise efficient chemotherapy of virus infections and cancer. In the genome of M. hyorhinis, a TP (thymidine phosphorylase) gene has been annotated. This gene was cloned, expressed in Escherichia coli and kinetically characterized. Whereas the mycoplasma TP efficiently catalyses the phosphorolysis of thymidine (Km=473 μM) and deoxyuridine (Km=578 μM), it prefers uridine (Km=92 μM) as a substrate. Our kinetic data and sequence analysis revealed that the annotated M. hyorhinis TP belongs to the NP (nucleoside phosphorylase)-II class PyNPs (pyrimidine NPs), and is distinct from the NP-II class TP and NP-I class UPs (uridine phosphorylases). M. hyorhinis PyNP also markedly differs from TP and UP in its substrate specificity towards therapeutic nucleoside analogues and susceptibility to clinically relevant drugs. Several kinetic properties of mycoplasma PyNP were explained by in silico analyses.

Cancer Cells Cut Homophilic Cell Adhesion Molecules and Run

The term contact inhibition (CI) encompasses the cellular changes that result in cessation of cell migration and of proliferation due to signals transduced when one cell comes into physical contact with another cell. Cancer cells, however, do not contact inhibit. A molecular understanding of the loss of CI in cancer cells is important for understanding tumor progression. In this Perspective, we propose that the loss of CI observed in cancer cells is the result of extracellular proteolysis of transmembrane cell-cell cell adhesion molecules (CAM) in the tumor microenvironment. Proteolysis of homophilic cell-cell CAMs results in a shed extracellular fragment and released cytoplasmic fragment(s) that disrupts adhesion and induces signals that promote proliferation and/or migration. The importance of this observation in tumor progression is supported by the presence of the shed extracellular fragments of homophilic cell-cell CAMs in serum and tumor tissue of cancer patients suggesting that instead of acting as tumor suppressors, the shed CAM extracellular and cytoplasmic fragments actually function as oncogenes. The study of cell-cell CAM cleavage will provide important and novel means of diagnosing, imaging, and treating tumor progression.