Discovery of a new marker to identify myeloid cells associated with metastatic breast tumours.

BACKGROUND: Myeloid cells play an essential role in cancer metastasis. The phenotypic diversity of these cells during cancer development has attracted great interest; however, their functional heterogeneity and plasticity have limited their role as prognostic markers and therapeutic targets. METHODS: To identify markers associated with myeloid cells in metastatic tumours, we compared transcriptomic data from immune cells sorted from metastatic and non-metastatic mammary tumours grown in BALB/cJ mice. To assess the translational relevance of our in vivo findings, we assessed human breast cancer biopsies and evaluated the association between arginase 1 protein expression in breast cancer tissues with tumour characteristics and patient outcomes. RESULTS: Among the differentially expressed genes, arginase 1 (ARG1) showed a unique expression pattern in tumour-infiltrating myeloid cells that correlated with the metastatic capacity of the tumour. Even though ARG1-positive cells were found almost exclusively inside the metastatic tumour, ARG1 protein was also present in the plasma. In human breast cancer biopsies, the presence of ARG1-positive cells was strongly correlated with high-grade proliferating tumours, poor prognosis, and low survival. CONCLUSION: Our findings highlight the potential use of ARG1-positive myeloid cells as an independent prognostic marker to evaluate the risk of metastasis in breast cancer patients.

Opposite and dynamic regulation of the interferon response in metastatic and non-metastatic breast cancer.

BACKGROUND: To our current understanding, solid tumors depend on suppressed local immune reactions, often elicited by the interaction between tumor cells and tumor microenvironment (TME) components. Despite an improved understanding of anti-cancer immune responses in the TME, it is still unclear how immuno-suppressive TME are formed and how some cancer cells survive and metastasize. METHODS: To identify the major adaptations that cancer cells undergo during tumor development and progression, we compared the transcriptome and proteome from metastatic 66cl4 and non-metastatic 67NR cell lines in culture versus their corresponding mouse mammary primary tumors. Using confocal microscopy, RT-qPCR, flow cytometry and western blotting, we studied the signaling pathway and the mechanisms involved. In addition, we used public gene expression data from human breast cancer biopsies to evaluate the correlation between gene expression and clinical outcomes in patients. RESULTS: We found that type I interferon (IFN-I) response was a key differentially regulated pathway between metastatic and non-metastatic cell lines and tumors. The IFN-I response was active in metastatic cancer cells in culture and markedly dampened when these cells formed primary tumors. Interestingly, the opposite was observed in non-metastatic cancer cells and tumors. Consistent with an active IFN-I response in culture, the metastatic cancer cells displayed elevated levels of cytosolic DNA from both mitochondria and ruptured micronuclei with concomitant activation of cGAS-STING signaling. Interestingly, decreased IFN-I-related gene expression in breast cancer biopsies correlated with an unfavourable prognosis in patients. CONCLUSION: Our findings show that IFN-I response is dampened in the tumors with the metastatic ability and lower IFN-I expression predicts poor prognosis in triple-negative and HER2 enriched breast cancer patients. This study highlights the possibility of reactivating the IFN-I response as a potential therapeutic strategy in breast cancer. Video Abstract.

NRF2 drives an oxidative stress response predictive of breast cancer.

Many breast cancer patients are diagnosed with small, well-differentiated, hormone receptor-positive tumors. Risk of relapse is not easily identified in these patients, resulting in overtreatment. To identify metastasis-related gene expression patterns, we compared the transcriptomes of the non-metastatic 67NR and metastatic 66cl4 cell lines from the murine 4T1 mammary tumor model. The transcription factor nuclear factor, erythroid 2-like 2 (NRF2, encoded by NFE2L2) was constitutively activated in the metastatic cells and tumors, and correspondingly a subset of established NRF2-regulated genes was also upregulated. Depletion of NRF2 increased basal levels of reactive oxygen species (ROS) and severely reduced ability to form primary tumors and lung metastases. Consistently, a set of NRF2-controlled genes was elevated in breast cancer biopsies. Sixteen of these were combined into a gene expression signature that significantly improves the PAM50 ROR score, and is an independent, strong predictor of prognosis, even in hormone receptor-positive tumors.

B cells from hyper-IgM patients carrying UNG mutations lack ability to remove uracil from ssDNA and have elevated genomic uracil.

The generation of high-affinity antibodies requires somatic hypermutation (SHM) and class switch recombination (CSR) at the immunoglobulin (Ig) locus. Both processes are triggered by activation-induced cytidine deaminase (AID) and require UNG-encoded uracil-DNA glycosylase. AID has been suggested to function as an mRNA editing deaminase or as a single-strand DNA deaminase. In the latter model, SHM may result from replicative incorporation of dAMP opposite U or from error-prone repair of U, whereas CSR may be triggered by strand breaks at abasic sites. Here, we demonstrate that extracts of UNG-proficient human B cell lines efficiently remove U from single-stranded DNA. In B cell lines from hyper-IgM patients carrying UNG mutations, the single-strand-specific uracil-DNA glycosylase, SMUG1, cannot complement this function. Moreover, the UNG mutations lead to increased accumulation of genomic uracil. One mutation results in an F251S substitution in the UNG catalytic domain. Although this UNG form was fully active and stable when expressed in Escherichia coli, it was mistargeted to mitochondria and degraded in mammalian cells. Our results may explain why SMUG1 cannot compensate the UNG2 deficiency in human B cells, and are fully consistent with the DNA deamination model that requires active nuclear UNG2. Based on our findings and recent information in the literature, we present an integrated model for the initiating steps in CSR.

ICU outcomes and survival in patients with severe COVID-19 in the largest health care system in central Florida.

BACKGROUND: Observational studies have consistently described poor clinical outcomes and increased ICU mortality in patients with severe coronavirus disease 2019 (COVID-19) who require mechanical ventilation (MV). Our study describes the clinical characteristics and outcomes of patients with severe COVID-19 admitted to ICU in the largest health care system in the state of Florida, United States. METHODS: Retrospective cohort study of patients admitted to ICU due to severe COVID-19 in AdventHealth health system in Orlando, Florida from March 11th until May 18th, 2020. Patients were characterized based on demographics, baseline comorbidities, severity of illness, medical management including experimental therapies, laboratory markers and ventilator parameters. Major clinical outcomes analyzed at the end of the study period were: hospital and ICU length of stay, MV-related mortality and overall hospital mortality of ICU patients. RESULTS: Out of total of 1283 patients with COVID-19, 131 (10.2%) met criteria for ICU admission (median age: 61 years [interquartile range (IQR), 49.5-71.5]; 35.1% female). Common comorbidities were hypertension (84; 64.1%), and diabetes (54; 41.2%). Of the 131 ICU patients, 109 (83.2%) required MV and 9 (6.9%) received ECMO. Lower positive end expiratory pressure (PEEP) were observed in survivors [9.2 (7.7-10.4)] vs non-survivors [10 (9.1-12.9] p = 0.004]. Compared to non-survivors, survivors had a longer MV length of stay (LOS) [14 (IQR 8-22) vs 8.5 (IQR 5-10.8) p< 0.001], Hospital LOS [21 (IQR 13-31) vs 10 (7-1) p< 0.001] and ICU LOS [14 (IQR 7-24) vs 9.5 (IQR 6-11), p < 0.001]. The overall hospital mortality and MV-related mortality were 19.8% and 23.8% respectively. After exclusion of hospitalized patients, the hospital and MV-related mortality rates were 21.6% and 26.5% respectively. CONCLUSIONS: Our study demonstrates an important improvement in mortality of patients with severe COVID-19 who required ICU admission and MV in comparison to previous observational reports and emphasizes the importance of standard of care measures in the management of COVID-19.

Extracts of proliferating and non-proliferating human cells display different base excision pathways and repair fidelity.

Base excision repair (BER) of damaged or inappropriate bases in DNA has been reported to take place by single nucleotide insertion or through incorporation of several nucleotides, termed short-patch and long-patch repair, respectively. We found that extracts from proliferating and non-proliferating cells both had capacity for single- and two-nucleotide insertion BER activity. However, patch size longer than two nucleotides was only detected in extracts from proliferating cells. Relative to extracts from proliferating cells, extracts from non-proliferating cells had approximately two-fold higher concentration of POLbeta, which contributed to most of two-nucleotide insertion BER. In contrast, two-nucleotide insertion in extracts from proliferating cells was not dependent on POLbeta. BER fidelity was two- to three-fold lower in extracts from the non-proliferating compared with extracts of proliferating cells. Furthermore, although one-nucleotide deletion was the predominant type of repair error in both extracts, the pattern of repair errors was somewhat different. These results establish two-nucleotide patch BER as a distinct POLbeta-dependent mechanism in non-proliferating cells and demonstrate that BER fidelity is lower in extracts from non-proliferating as compared with proliferating cells.

Autocrine activin A signalling in ovarian cancer cells regulates secretion of interleukin 6, autophagy, and cachexia.

BACKGROUND: The majority of patients with advanced cancer develop cachexia, a weight loss syndrome that severely reduces quality of life and limits survival. Our understanding of the underlying mechanisms that cause the condition is limited, and there are currently no treatment options that can completely reverse cachexia. Several tumour-derived factors and inflammatory mediators have been suggested to contribute to weight loss in cachectic patients. However, inconsistencies between studies are recurrent. Activin A and interleukin 6 (IL-6) are among the best studied factors that seem to be important, and several studies support their individual role in cachexia development. METHODS: We investigated the interplay between activin A and IL-6 in the cachexia-inducing TOV21G cell line, both in culture and in tumours in mice. We previously found that the human TOV21G cells secrete IL-6 that induces autophagy in reporter cells and cachexia in mice. Using this established cachexia cell model, we targeted autocrine activin A by genetic, chemical, and biological approaches. The secretion of IL-6 from the cancer cells was determined in both culture and tumour-bearing mice by a species-specific ELISA. Autophagy reporter cells were used to monitor the culture medium for autophagy-inducing activities, and muscle mass changes were evaluated in tumour-bearing mice. RESULTS: We show that activin A acts in an autocrine manner to promote the synthesis and secretion of IL-6 from cancer cells. By inhibiting activin A signalling, the production of IL-6 from the cancer cells is reduced by 40-50% (up to 42% reduction on protein level, P = 0.0048, and 48% reduction on mRNA level, P = 0.0308). Significantly reduced IL-6 secretion (P < 0.05) from the cancer cells is consistently observed when using biological, chemical, and genetic approaches to interfere with the autocrine activin A loop. Inhibiting activin signalling also reduces the ability of the cancer cells to accelerate autophagy in non-cancerous cells (up to 43% reduced autophagy flux, P = 0.0006). Coherent to the in vitro data, the use of an anti-activin receptor 2 antibody in cachectic tumour-bearing mice reduces serum levels of cancer cell-derived IL-6 by 62% (from 417 to 159 pg/mL, P = 0.03), and, importantly, it reverses cachexia and counteracts loss of all measured muscle groups (P < 0.0005). CONCLUSIONS: Our data support a functional link between activin A and IL-6 signalling pathways and indicate that interference with activin A-induced IL-6 secretion from the tumour has therapeutic potential for cancer-induced cachexia.

Cancer cachexia associates with a systemic autophagy-inducing activity mimicked by cancer cell-derived IL-6 trans-signaling.

The majority of cancer patients with advanced disease experience weight loss, including loss of lean body mass. Severe weight loss is characteristic for cancer cachexia, a condition that significantly impairs functional status and survival. The underlying causes of cachexia are incompletely understood, and currently no therapeutic approach can completely reverse the condition. Autophagy coordinates lysosomal destruction of cytosolic constituents and is systemically induced by starvation. We hypothesized that starvation-mimicking signaling compounds secreted from tumor cells may cause a systemic acceleration of autophagy during cachexia. We found that IL-6 secreted by tumor cells accelerates autophagy in myotubes when complexed with soluble IL-6 receptor (trans-signaling). In lung cancer patients, were cachexia is prevalent, there was a significant correlation between elevated IL-6 expression in the tumor and poor prognosis of the patients. We found evidence for an autophagy-inducing bioactivity in serum from cancer patients and that this is clearly associated with weight loss. Importantly, the autophagy-inducing bioactivity was reduced by interference with IL-6 trans-signaling. Together, our findings suggest that IL-6 trans-signaling may be targeted in cancer cachexia.

Monoclonal B-cell hyperplasia and leukocyte imbalance precede development of B-cell malignancies in uracil-DNA glycosylase deficient mice.

Ung-deficient mice have reduced class switch recombination, skewed somatic hypermutation, lymphatic hyperplasia and a 22-fold increased risk of developing B-cell lymphomas. We find that lymphomas are of follicular (FL) and diffuse large B-cell type (DLBCL). All FLs and 75% of the DLBCLs were monoclonal while 25% were biclonal. Monoclonality was also observed in hyperplasia, and could represent an early stage of lymphoma development. Lymphoid hyperplasia occurs very early in otherwise healthy Ung-deficient mice, observed as a significant increase of splenic B-cells. Furthermore, loss of Ung also causes a significant reduction of T-helper cells, and 50% of the young Ung(-/-) mice investigated have no detectable NK/NKT-cell population in their spleen. The immunological imbalance is confirmed in experiments with spleen cells where the production of the cytokines interferon gamma, interleukin 6 and interleukin 2 is clearly different in wild type and in Ung-deficient mice. This suggests that Ung-proteins, directly or indirectly, have important functions in the immune system, not only in the process of antibody maturation, but also for production and functions of immunologically important cell types. The immunological imbalances shown here in the Ung-deficient mice may be central in the development of lymphomas in a background of generalised lymphoid hyperplasia.

Gene-targeted mice lacking the Ung uracil-DNA glycosylase develop B-cell lymphomas.

Mice deficient in the Ung uracil-DNA glycosylase have an increased level of uracil in their genome, consistent with a major role of Ung counteracting U:A base pairs arising by misincorporation of dUMP during DNA replication. A complementary uracil-excising activity apparently acts on premutagenic U:G lesions resulting from deamination of cytosine throughout the genome. However, Ung specifically processes U:G lesions targeted to immunoglobulin variable (V) genes during somatic hypermutation and class-switch recombination. Gene-targeted Ung(-/-) null mice remained tumour-free and showed no overt pathological phenotype up to approximately 12 months of age. We have monitored a large cohort of ageing Ung(-/-) mice and, beyond 18 months of age, they had a higher morbidity than Ung(+/+) controls. Post-mortem analyses revealed pathological changes in lymphoid organs, abnormal lymphoproliferation, and a greatly increased incidence of B-cell lymphomas in older Ung-deficient mice. These are the first data reporting the development of spontaneous malignancies in mice due to deficiency in a DNA glycosylase. Furthermore, they support a specific role for Ung in the immune system, with lymphomagenesis being related to perturbed processing of antibody genes in germinal centre B cells.

Trypanosoma cruzi contains a single detectable uracil-DNA glycosylase and repairs uracil exclusively via short patch base excision repair.

Enzymes involved in genomic maintenance of human parasites are attractive targets for parasite-specific drugs. The parasitic protozoan Trypanosoma cruzi contains at least two enzymes involved in the protection against potentially mutagenic uracil, a deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and a uracil-DNA glycosylase belonging to the highly conserved UNG-family. Uracil-DNA glycosylase activities excise uracil from DNA and initiate a multistep base-excision repair (BER) pathway to restore the correct nucleotide sequence. Here we report the biochemical characterisation of T.cruzi UNG (TcUNG) and its contribution to the total uracil repair activity in T.cruzi. TcUNG is shown to be the major uracil-DNA glycosylase in T.cruzi. The purified recombinant TcUNG exhibits substrate preference for removal of uracil in the order ssU>U:G>U:A, and has no associated thymine-DNA glycosylase activity. T.cruzi apparently repairs U:G DNA substrate exclusively via short-patch BER, but the DNA polymerase involved surprisingly displays a vertebrate POLdelta-like pattern of inhibition. Back-up UDG activities such as SMUG, TDG and MBD4 were not found, underlying the importance of the TcUNG enzyme in protection against uracil in DNA and as a potential target for drug therapy.

The N-terminally truncated µ3 and µ3-like opioid receptors are transcribed from a novel promoter upstream of exon 2 in the human OPRM1 gene.

The human µ opioid receptor gene, OPRM1, produces a multitude of alternatively spliced transcripts encoding full-length or truncated receptor variants with distinct pharmacological properties. The majority of these transcripts are transcribed from the main promoter upstream of exon 1, or from alternate promoters associated with exons 11 and 13. Two distinct transcripts encoding six transmembrane domain (6TM) hMOR receptors, µ3 and µ3-like, have been reported, both starting with the first nucleotide in exon 2. However, no mechanism explaining their initiation at exon 2 has been presented. Here we have used RT-PCR with RNA from human brain tissues to demonstrate that the µ3 and µ3-like transcripts contain nucleotide sequences from the intron 1-exon 2 boundary and are transcribed from a novel promoter located upstream of exon 2. Reporter gene assays confirmed the ability of the novel promoter to drive transcription in human cells, albeit at low levels. We also report the identification of a "full-length" seven transmembrane domain (7TM) version of µ3, hMOR-1A2, which also contains exon 1, and a novel transcript, hMOR-1Y2, with the potential to encode the previously reported hMOR-1Y receptor, but with exon Y spliced to exon 4 instead of exon 5 as in hMOR-1Y. Heterologous expression of GFP-tagged hMOR variants in HEK 293 cells showed that both 6TM receptors were retained in the intracellular compartment and were unresponsive to exogenous opioid exposure as assessed by their ability to redistribute or affect cellular cAMP production, or to promote intracellular Ca(2+) release. Co-staining with an antibody specific for endoplasmic reticulum (ER) indicated that the µ3-like receptor was retained at the ER after synthesis. 7TM receptors hMOR-1A2 and hMOR-1Y2 resided in the plasma membrane, and were responsive to opioids. Notably, hMOR-1A2 exhibits novel functional properties in that it did not internalize in response to the opioid peptide [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO).

XRCC1 coordinates disparate responses and multiprotein repair complexes depending on the nature and context of the DNA damage.

XRCC1 is a scaffold protein capable of interacting with several DNA repair proteins. Here we provide evidence for the presence of XRCC1 in different complexes of sizes from 200 to 1500 kDa, and we show that immunoprecipitates using XRCC1 as bait are capable of complete repair of AP sites via both short patch (SP) and long patch (LP) base excision repair (BER). We show that POLß and PNK colocalize with XRCC1 in replication foci and that POLß and PNK, but not PCNA, colocalize with constitutively present XRCC1-foci as well as damage-induced foci when low doses of a DNA-damaging agent are applied. We demonstrate that the laser dose used for introducing DNA damage determines the repertoire of DNA repair proteins recruited. Furthermore, we demonstrate that recruitment of POLß and PNK to regions irradiated with low laser dose requires XRCC1 and that inhibition of PARylation by PARP-inhibitors only slightly reduces the recruitment of XRCC1, PNK, or POLß to sites of DNA damage. Recruitment of PCNA and FEN-1 requires higher doses of irradiation and is enhanced by XRCC1, as well as by accumulation of PARP-1 at the site of DNA damage. These data improve our understanding of recruitment of BER proteins to sites of DNA damage and provide evidence for a role of XRCC1 in the organization of BER into multiprotein complexes of different sizes.

Variation in the COMT gene: implications for pain perception and pain treatment.

Catechol-O-methyltransferase (COMT) is an enzyme that inactivates biologically-active catechols, including the important neurotransmitters dopamine, noradrenaline and adrenaline. These neurotransmitters are involved in numerous physiological processes, including modulation of pain. Genetic variation in the COMT gene has been implicated in variable response to various experimental painful stimuli, variable susceptibility to develop common pain conditions, as well as the variable need for opioids in the treatment of cancer pain. Increased insight into how genetic variants within the COMT locus affect pain perception will contribute to improved understanding of the mechanisms involved in the development of common human pain disorders and may lead to improved strategies for pain treatment. So far, a remarkable complex relationship between COMT genotypes or haplotypes and pain phenotypes has been revealed.

Incorporation of dUMP into DNA is a major source of spontaneous DNA damage, while excision of uracil is not required for cytotoxicity of fluoropyrimidines in mouse embryonic fibroblasts.

Uracil may arise in DNA as a result of deamination of cytosine or through incorporation of dUMP instead of dTMP during replication. We have studied the steady-state levels of uracil in the DNA of primary cells and mouse embryonic fibroblast (MEF) cell lines from mice deficient in the Ung uracil-DNA glycosylase. The results show that the levels of uracil in the DNA of Ung(-/-) cells strongly depend on proliferation, indicating that the uracil residues originate predominantly from misincorporation during replication. Treatment with 5-fluoro-2'-deoxyuridine (5-FdUrd) or 5-fluorouracil (5-FU) gives rise to a dose-dependent increase of uracil in Ung(-/-) MEFs (up to 1.5-fold) but not in wild-type cells. Interestingly, Ung(-/-) MEFs accumulate AP-sites as well as uracil in response to 5-FdUrd but not to 5-FU. This accumulation of repair intermediates suggests a loss of tightly co-ordinated repair in the absence of Ung, and correlates with stronger inhibition of cell proliferation in response to 5-FdUrd, but not to 5-FU, in Ung(-/-) MEFs compared with wild-type cells. However, other cytotoxic effects of these fluoropyrimidines are comparable in both wild-type and Ung-deficient cells, demonstrating that excision of uracil from DNA by the Ung uracil-DNA glycosylase is not a prerequisite for obtaining cytotoxicity.