Targeting Efferocytosis in Inflammaging.

Rapid removal of apoptotic cells by phagocytes, a process known as efferocytosis, is key for the maintenance of tissue homeostasis, the resolution of inflammation, and tissue repair. However, impaired efferocytosis can result in the accumulation of apoptotic cells, subsequently triggering sterile inflammation through the release of endogenous factors such as DNA and nuclear proteins from membrane permeabilized dying cells. Here, we review the molecular basis of the three key phases of efferocytosis, that is, the detection, uptake, and degradation of apoptotic materials by phagocytes. We also discuss how defects in efferocytosis due to the alteration of phagocytes and dying cells can contribute to the low-grade chronic inflammation that occurs during aging, described as inflammaging. Lastly, we explore opportunities in targeting and harnessing the efferocytic machinery to limit aging-associated inflammatory diseases.

Caspase-8-driven apoptotic and pyroptotic crosstalk causes cell death and IL-1ß release in X-linked inhibitor of apoptosis (XIAP) deficiency.

Genetic lesions in X-linked inhibitor of apoptosis (XIAP) pre-dispose humans to cell death-associated inflammatory diseases, although the underlying mechanisms remain unclear. Here, we report that two patients with XIAP deficiency-associated inflammatory bowel disease display increased inflammatory IL-1ß maturation as well as cell death-associated caspase-8 and Gasdermin D (GSDMD) processing in diseased tissue, which is reduced upon patient treatment. Loss of XIAP leads to caspase-8-driven cell death and bioactive IL-1ß release that is only abrogated by combined deletion of the apoptotic and pyroptotic cell death machinery. Namely, extrinsic apoptotic caspase-8 promotes pyroptotic GSDMD processing that kills macrophages lacking both inflammasome and apoptosis signalling components (caspase-1, -3, -7, -11 and BID), while caspase-8 can still cause cell death in the absence of both GSDMD and GSDME when caspase-3 and caspase-7 are present. Neither caspase-3 and caspase-7-mediated activation of the pannexin-1 channel, or GSDMD loss, prevented NLRP3 inflammasome assembly and consequent caspase-1 and IL-1ß maturation downstream of XIAP inhibition and caspase-8 activation, even though the pannexin-1 channel was required for NLRP3 triggering upon mitochondrial apoptosis. These findings uncouple the mechanisms of cell death and NLRP3 activation resulting from extrinsic and intrinsic apoptosis signalling, reveal how XIAP loss can co-opt dual cell death programs, and uncover strategies for targeting the cell death and inflammatory pathways that result from XIAP deficiency.

Spatial Distribution and Densities of CD103+ and FoxP3+ Tumor Infiltrating Lymphocytes by Digital Analysis for Outcome Prediction in Breast Cancer.

BACKGROUND: The evaluation of tumor-infiltrating lymphocytes (TILs) for breast cancer prognosis is now established. However, the clinical value for their spatial distributions of specific immune subsets, namely CD103+ tissue-resident memory T cells FoxP3+ regulatory T ells, have not been thoroughly examined. METHOD: Representative whole sections of breast cancers were subjected to CD103 and FoxP3 double staining. Their density, ratio, and spatial features were analyzed in tumor area and tumor-stromal interface. Their associations with clinicopathological parameters and patient's prognosis were analyzed. RESULTS: CD103 TILs were closer to tumor nests than FoxP3 TILs in the tumor-stromal interface. Their densities were associated with high-grade disease, TNBC, and stromal TILs. High stromal FoxP3 (sFoxP3) TILs and close proximity of sCD103 TILs to tumor were independently associated with better survival at multivariate analysis. Subgroup analysis showed the high FoxP3 TILs density associated better survival was seen in HER2-OE and TNBC subtypes while the proximity of CD103 TILs to tumor nests associated better survival was seen in luminal cancers. CONCLUSION: The prognostic impact of CD103 and FoxP3 TILs in breast cancer depends on their spatial localization. High sFoxP3 TIL density and the lower distance of CD103 TILs from the tumor nests had independent favorable prognostic values.

Comparison and review of abrasive bronchial brushing versus non-abrasive aspiration, lavage and washing - Higher sensitivity but with risk of over-diagnosis for bronchial brushing.

Bronchial exfoliative cytology is classified as non-abrasive (washing, aspiration and bronchoalveolar lavage) and abrasive (brushing). Brush abrasion dislodges epithelial cells but can induce bleeding and cytomorphologic artifacts. In this study, the largest cohort to date of bronchial cytology specimens were referenced against bronchial biopsy as the reference standard. Findings in the study will be useful for selecting biopsy modality and reducing necessary procedural risks. All consecutive bronchial cytology and bronchial biopsy from 1995 to 2022 were retrieved. The diagnoses were reviewed and categorized into five-tiered diagnostic categories to compare diagnostic agreement and concordance. Review of 14,148 specimens yielded 3963 non-abrasive, 2378 abrasive cytology specimens matched to biopsy, with 4355 matches between non-abrasive and abrasive cytology specimens. Agreement between non-abrasive and abrasive cytology was moderate (κ = 0.580), and similar when referenced against biopsy (κ = 0.456 (non-abrasive), κ = 0.498 (abrasive)). Abrasive bronchial cytology showed a higher percentage of malignant diagnosis (20.95 % vs. 12.63 %, p < 0.001) and over-diagnosis rate (36.40 % vs. 29.79 %, p < 0.001), but higher sensitivity (0.747 vs. 0.572, p = 0.002). For subgroup analysis of transbronchial biopsies, matched abrasive cytology showed higher discordant rates (p < 0.05) and lower accuracy (0.907 vs. 0.873, p = 0.020). With the added bleeding risk associated with brushing, abrasive techniques may only be preferable in cases with clinical or bronchoscopic suspicion of malignancy, in particular endobronchial mucosal lesions. For routine bronchoscopy, non-abrasive bronchial cytology appears to be adequate.

Anatomical site and size of sentinel lymph node metastasis predicted additional axillary tumour burden and breast cancer survival.

AIMS: Sentinel lymph node (SLN) biopsy is the current standard assessment for tumour burden in axillary lymph node (ALN). However, not all SLN+ patients have ALN metastasis. The prognostic implication of SLN features is not clear. We aimed to evaluate predictive factors for ALN metastasis and the clinical value of SLN features. METHODS AND RESULTS: A total of 228 SLN+ and 228 SLN- (with matched year and grade) cases were included. Clinicopathological features in SLN, ALN and primary tumours, treatment data and survival data were analysed according to ALN status and outcome. Except for larger tumour size and the presence of LVI (both P < 0.001), no significant differences were found in SLN- and SLN+ cases. Only 31.8% of SLN+ cases with ALN dissection had ALN metastasis. The presence of macrometastases (MaM), extranodal extension (ENE), deeper level of tumour invasion in SLN and more SLN+ nodes were associated with ALN metastasis (P ≤ 0.025). Moreover, isolated tumour cells (ITC) and level of tumour invasion in SLN were independent adverse prognostic features for disease-free survival and breast cancer-specific survival, respectively. Interestingly, cases with ITC located in the subcapsular region have better survival than those in cortex (OS: χ2  = 4.046, P = 0.044). CONCLUSIONS: Our study identified features in SLN, i.e. the level of tumour invasion at SLN and tumour size in SLN as useful predictors for both ALN metastasis and breast cancer outcome. The presence of ITC, particularly those with a deeper invasion in SLN, portended a worse prognosis. Proper attention should be taken for their management.

Human MAIT cell cytolytic effector proteins synergize to overcome carbapenem resistance in Escherichia coli.

Mucosa-associated invariant T (MAIT) cells are abundant antimicrobial T cells in humans and recognize antigens derived from the microbial riboflavin biosynthetic pathway presented by the MHC-Ib-related protein (MR1). However, the mechanisms responsible for MAIT cell antimicrobial activity are not fully understood, and the efficacy of these mechanisms against antibiotic resistant bacteria has not been explored. Here, we show that MAIT cells mediate MR1-restricted antimicrobial activity against Escherichia coli clinical strains in a manner dependent on the activity of cytolytic proteins but independent of production of pro-inflammatory cytokines or induction of apoptosis in infected cells. The combined action of the pore-forming antimicrobial protein granulysin and the serine protease granzyme B released in response to T cell receptor (TCR)-mediated recognition of MR1-presented antigen is essential to mediate control against both cell-associated and free-living, extracellular forms of E. coli. Furthermore, MAIT cell-mediated bacterial control extends to multidrug-resistant E. coli primary clinical isolates additionally resistant to carbapenems, a class of last resort antibiotics. Notably, high levels of granulysin and granzyme B in the MAIT cell secretomes directly damage bacterial cells by increasing their permeability, rendering initially resistant E. coli susceptible to the bactericidal activity of carbapenems. These findings define the role of cytolytic effector proteins in MAIT cell-mediated antimicrobial activity and indicate that granulysin and granzyme B synergize to restore carbapenem bactericidal activity and overcome carbapenem resistance in E. coli.

Combining Analysis of Tumor-infiltrating Lymphocytes (TIL) and PD-L1 Refined the Prognostication of Breast Cancer Subtypes.

BACKGROUND: PD-L1 has been used as a biomarker to select patients for treatment of PD-1/PD-L1 inhibitors. MATERIALS AND METHODS: In this study, we assessed the clinicopathological features of breast cancers that are associated with PD-L1 expression, as well as its relationship with other immune components and its prognostic significance. RESULTS: Totally 1752 cases were included in this cohort. PD-L1 expression in tumor-infiltrating immune cells (PD-L1-IC) expression and in tumor cells (PD-L1-TC) expression were identified in 34.2% and 10.1% of cases, respectively, and they showed a positive correlation with higher tumor grade, morphological apocrine features, presence of necrosis, and higher stromal tumor-infiltrating lymphocytes (sTIL). PD-L1-IC and PD-L1-TC expression correlated positively with each other, and both of them were negatively associated with estrogen receptor and progesterone receptor and positively associated with Ki67, HER2, EGFR, p63, and p-cadherin. In survival analysis, PD-L1-IC expression was associated with better disease-free survival (DFS) and breast cancer-specific survival (BCSS) in HER2-overexpressed (HER2-OE) cancers and high-grade luminal B cancers. In triple-negative breast cancers (TNBC) and HER2-OE cancers, compared with sTIL low PD-L1-IC negative cases, sTIL high cases showed significantly better DFS independent of PD-L1-IC status. sTIL low PD-L1-IC positive cases also demonstrated a better DFS in HER2-OE cancers. In high-grade luminal B cancers, sTIL high PD-L1-IC positive cases showed the best BCSS. CONCLUSION: The data suggested that the combining analysis of sTIL and PD-L1-IC expression refined the prognostication of breast cancer subtypes. Cases with high TIL and PD-LI-IC expression appear to be more immune active.

Apoptotic Bodies: Mechanism of Formation, Isolation and Functional Relevance.

In the final stages of apoptosis, apoptotic cells can generate a variety of membrane-bound vesicles known as apoptotic extracellular vesicles (ApoEVs). Apoptotic bodies (ApoBDs), a major subset of ApoEVs, are formed through a process termed apoptotic cell disassembly characterised by a series of tightly regulated morphological steps including plasma membrane blebbing, apoptotic membrane protrusion formation and fragmentation into ApoBDs. To better characterise the properties of ApoBDs and elucidate their function, a number of methods including differential centrifugation, filtration and fluorescence-activated cell sorting were developed to isolate ApoBDs. Furthermore, it has become increasingly clear that ApoBD formation can contribute to various biological processes such as apoptotic cell clearance and intercellular communication. Together, recent literature demonstrates that apoptotic cell disassembly and thus, ApoBD formation, is an important process downstream of apoptotic cell death. In this chapter, we discuss the current understandings of the molecular mechanisms involved in regulating apoptotic cell disassembly, techniques for ApoBD isolation, and the functional roles of ApoBDs in physiological and pathological settings.

Fine needle aspiration cytology of metastatic carcinomas with papillary architecture: A systemic assessment of clinical, cytological and immunohistochemical parameters.

BACKGROUND: Papillary structures are frequently encountered in metastatic carcinomas from various organs and tumours of different histotypes. This study aims to investigate the predictive value of fine needle aspiration cytology (FNAC), immunohistochemistry (IHC) and the clinical parameters which can be examined in the assessment of the primary sites of metastatic carcinomas with papillary architecture. METHODS: FNAC samples of metastatic carcinomas with papillary architecture were evaluated for overall cellularity, epithelial cohesion, background features, papillary architecture, cytology and IHC. The corresponding clinical information was also reviewed. RESULTS: A total of 130 cases were included. The most common primary sites were thyroid (38.5%), lung (30.8%) and gynecological organs (22.3%); the others were pancreaticobiliary, urothelial, colorectal, and esophageal. Age (P = 0.039), biopsy site (P < 0.001) and laterality (P = 0.006) correlated with primary site. Papillary structures were confirmed on biopsy/excision of most cases (n = 85/87, 97.7%). Thyroid primaries exhibited broad papillary stalks, thin lining epithelium, fewer epithelial polymorphs, and the presence of background giant cells and histiocytes (P = 0.021- < 0.001). Low-grade cytological features, nuclear grooves and inclusions (P < 0.001) were seen in thyroid primaries. High-grade features (P < 0.001-0.49), multinucleated tumour cells, apoptotic bodies and mitoses (P < 0.001-0.49) were more common in lung/gynecological primaries. Multivariate analysis identified nuclear/cytoplasmic ratio, chromatin character, the presence of nuclear grooves and mitosis as independent features (P = 0.001-0.024). TTF1/TGB/PAX8 panel results showed good agreement with the cytological assessment and site of primary. CONCLUSION: Papillary structures and cytological features are reproducible in FNAC assessment of metastases and their corresponding primary sites. Cytological features, IHC and clinical information are invaluable in determining the primary site.

Novel Formulation of Undecylenic Acid induces Tumor Cell Apoptosis.

Undecylenic acid, a monounsaturated fatty acid, is currently in clinical use as a topical antifungal agent, however the potential for therapeutic application in other disease settings has not been investigated. In this study, we describe a novel platform for the solubilization of fatty acids using amino acids and utilize this approach to define a tumoricidal activity and underlying mechanism for undecylenic acid. We examined a novel formulation of undecylenic acid compounded with L-Arginine, called GS-1, that induced concentration-dependent tumor cell death, with undecylenic acid being the cytotoxic component. Further investigation revealed that GS-1-mediated cell death was caspase-dependent with a reduction in mitochondrial membrane potential, suggesting a pro-apoptotic mechanism of action. Additionally, GS-1 was found to localize intracellularly to lipid droplets. In contrast to previous studies where lipid droplets have been shown to be protective against fatty acid-induced cell death, we showed that lipid droplets could not protect against GS-1-induced cytotoxicity. We also found a role for Fatty Acid Transport Protein 2 (FATP2) in the uptake of this compound. Collectively, this study demonstrates that GS-1 has effective pro-apoptotic antitumor activity in vitro and, together with the novel platform of fatty acid solubilization, contributes to the re-emerging field of fatty acids as potential anti-cancer therapeutics.

SETD2 alterations and histone H3K36 trimethylation in phyllodes tumor of breast.

PURPOSE: SETD2 is one of the key epigenetic regulatory genes involved in histone modifications. Its alterations were potentially oncogenic and commonly found in cancers. Interestingly, SETD2 is one of the most frequent mutated genes found exclusively in phyllodes tumor of the breast (PT). However, little has been done to further characterize SETD2 alterations in PT. METHODS: In this study, we examined the alterations of SETD2 gene and protein expression in a large cohort of PTs. Their correlations with SETD2 downstream target, H3K36me3 expression, and clinicopathologic features in PT were also assessed. RESULTS: SETD2 mutation was found in 15.9% of our cases and was mostly predicted to be damaging mutations. Interestingly, SETD2 mutations were associated with lower H3K36me3 expression, particularly those with damaging mutations (p = .041). Neither SETD2 mutations nor H3K36me3 expression was associated with PT grading and other clinicopathological features. By contrast, the SETD2 protein expression cannot reflect its mutation status and showed a different trend of clinicopathological correlations from H3K36me3. CONCLUSIONS: Our findings may suggest a potential involvement of epigenetic regulation via SETD2 alterations and downstream H3K36me3 on PT development. SETD2 mutations may occur early in the pathogenic process of PTs and its loss per se may not be sufficient for progression to malignancy. Exclusive alterations of SETD2 in PT can be used as markers for the diagnosis of fibroepithelial lesions. The association of H3K36me3 with SETD2 mutations may also indicate the value of evaluation of H3K36me3 expression in the diagnosis of fibroepithelial lesions.

The significance of highlighting the oestrogen receptor low category in breast cancer.

The latest ASCO/CAP guideline has recommended to report oestrogen receptor (ER) low cases (ERlo; 1-10%) as "ER low positive category", prompting us to compare the clinicopathologic features, biomarkers, survival and treatment of the ERlo cases with other subgroups (ER negative (ERneg) and ER high (ERhi)). ERlo cases revealed more similar clinicopathologic and biomarker profiles (including younger age, larger tumour, high proliferation, HER2 and basal markers expression) to ERneg than ERhi cancers. The ERlo cases receiving hormonal therapy showed a similarly poor outcome as ERneg cancers. However, majority of ERlo cases were downstaged to stage I in the 8th AJCC pathological prognostic staging, highlighting a risk of potential under treatment. Overall, our data highlighted the differences of ERlo from other ERpos cases and their management should be considered separately.

Smac mimetics can provoke lytic cell death that is neither apoptotic nor necroptotic.

Smac mimetics, or IAP antagonists, are a class of drugs currently being evaluated as anti-cancer therapeutics. These agents antagonize IAP proteins, including cIAP1/2 and XIAP, to induce cell death via apoptotic or, upon caspase-8 deficiency, necroptotic cell death pathways. Many cancer cells are unresponsive to Smac mimetic treatment as a single agent but can be sensitized to killing in the presence of the cytokine TNFα, provided either exogenously or via autocrine production. We found that high concentrations of a subset of Smac mimetics could provoke death in cells that did not produce TNFα, despite sensitization at lower concentrations by TNFα. The ability of these drugs to kill did not correlate with valency. These cells remained responsive to the lethal effects of Smac mimetics at high concentrations despite genetic or pharmacological impairments in apoptotic, necroptotic, pyroptotic, autophagic and ferroptotic cell death pathways. Analysis of dying cells revealed necrotic morphology, which was accompanied by the release of lactate dehydrogenase and cell membrane rupture without prior phosphatidylserine exposure implying cell lysis, which occurred over a several hours. Our study reveals that cells incapable of autocrine TNFα production are sensitive to some Smac mimetic compounds when used at high concentrations, and this exposure elicits a lytic cell death phenotype that occurs via a mechanism not requiring apoptotic caspases or necroptotic effectors RIPK3 or MLKL. These data reveal the possibility that non-canonical cell death pathways can be triggered by these drugs when applied at high concentrations.

The Clinical Significance of Neuroendocrine Features in Invasive Breast Carcinomas.

The latest World Health Organization (WHO) classification categorized invasive breast carcinomas (IBCs) with neuroendocrine (NE) differentiations into neuroendocrine neoplasms (including well-differentiated neuroendocrine tumor [NET] and poorly differentiated neuroendocrine carcinoma [NEC]) and IBC no special type with NE features (IBC-NST-NE). However, little is documented of the clinical significance of this classification; also the precise thresholds and choices of NE markers were variable. In the current study, a large cohort of patients with IBC with NE differentiation were morphologically classified based on the WHO criteria and the clinical relevance of expression of different NE markers (synaptophysin [SYN], chromogranin [CG], and CD56) was evaluated. Among 1,372 IBCs, 52 NET (3.8%) and 172 IBC-NST-NE (12.5%) were identified. Compared with the IBC-no NE cases, NET and IBC-NST-NE were similarly associated with positive estrogen receptor (ER) expression and lower grade (p < .001). For patient outcome, IBC-NST-NE, but not NET, demonstrated significantly worse survival than the IBC-no NE cases. Based on high (≥50%) and low (<50%) expression for each NE marker, independent poor disease-free survival for SYNlo CGlo and SYNhi CGlo cancers (IBC-no NE cases as references, hazard ratio [HR], ≤1.429; p ≤ .026) was found. Interestingly, SYN and CG expression correlated with each other and they shared similar clinicopathologic characteristics; but not with with CD56. In addition, CD56-only positive cases were associated with hormone receptors negativity and basal markers positivity (p ≤ .019), and patients' outcome was similar to IBC-no NE cancers. Our findings suggested that NE markers expression may provide information to fine tune treatment strategy. The relevance of CD56 as NE marker requires further studies. IMPLICATIONS FOR PRACTICE: Invasive breast carcinomas (IBCs) with neuroendocrine (NE) differentiation are heterogeneous in clinicopathologic parameters, biomarker expression, and prognosis. However, there are no specific therapies targeting NE differentiation, and all carcinomas with any NE differentiation are treated similarly as other IBCs. The results of this study suggest that stratification based on NE marker expression levels may provide added prognostically pertinent information, aiding better treatment strategy. In addition, CD56-only positive carcinomas showed a different clinicopathologic and biomarker expression profile compared with those with chromogranin and synaptophysin expression. Relevance of CD56 as an NE marker requires further studies.

Co-expression of HLA-I loci improved prognostication in HER2+ breast cancers.

The underlying basis for cancer immune evasion is important for effective immunotherapy and prognosis in breast cancers. Human leucocyte antigens (HLA)-I comprising three classical antigens (HLA-A, -B and -C) is mandatory for anti-tumor immunity. Its loss occurred frequently in many cancers resulting in effective immune evasion. Most studies examined HLA-I as a whole. Alterations in specific locus could have different clinical ramifications. Hence, we evaluated the expression of the three HLA-I loci in a large cohort of breast cancers. Low expression of HLA-A, -B and -C were found in 71.1%, 66.3%, and 60.2% of the cases. Low and high expression in all loci was found in 48.3% and 17.9% of the cases respectively. The remaining showed high expression in one or two loci. Cases with all HLA high expression (all HLA high) was frequent in the ER-HER2- (27.4%) and ER-HER2+ (23.1%) cases and was associated with characteristic pathologic features related to these tumor (higher grade, necrosis, high tumor infiltrating lymphocyte (TIL), pT stage, low hormonal receptor, high basal marker expression) (p ≤ 0.019). Interestingly, in HER2+ cancers, only cases with all HLA high and high TIL showed significantly better survival. In node positive cancers, concordant high HLA expression in primary tumors and nodal metastases was favorable prognostically (DFS: HR = 0.741, p < 0.001; BCSS: HR = 0.699, p = 0.003). The data suggested an important clinical value of a combined analysis on the co-expression HLA-I status in both primary and metastatic tumors. This could be a potential additional key component to be incorporated into TIL evaluation for improved prognostication.

Combined SOX10 GATA3 is most sensitive in detecting primary and metastatic breast cancers: a comparative study of breast markers in multiple tumors.

PURPOSE: For invasive breast cancer (IBC), high SOX10 expression was reported particularly in TNBC. This raised the possibility that SOX10 may complement other breast markers for determining cancers of breast origin. METHODS: Here, we compared the expression of SOX10 with other breast markers (GATA3, mammaglobin and GCDFP15) and their combined expression in a large cohort of IBC together with nodal metastases. We have also evaluated the expression of GATA3 and SOX10 in a wide spectrum of non-breast carcinomas to assess their value as breast specific markers. RESULTS: Compared with other markers, SOX10 showed lower overall sensitivity (6.5%), but higher sensitivity in TNBC (31.4%) than other breast markers including GATA3 (29.7% for TNBC). Its expression demonstrated the highest concordance between the paired IBC and nodal metastases (96.4%, κ = 0.663) among all the breast markers. More importantly, SOX10 identified many GATA3-negative TNBC, thus the SOX10/GATA3 combination was the most sensitive marker combination for IBC (86.6%). For non-breast carcinoma, a high SOX10/GATA3 expression rate was found in melanoma (77.9%, predominately expressed SOX10), urothelial carcinoma (82.0%, predominately expressed GATA3) and salivary gland tumors (69.4%). Other carcinomas, including cancers from lungs, showed very low expression for the marker combination. CONCLUSIONS: The data suggested that SOX10/GATA3 combination can be used for differentiating metastases of breast and multiple non-breast origins. However, the differentiation with melanoma and urothelial tumors required more careful histologic examination, thorough clinical information and additional site-specific IHC markers. For salivary gland tumors, the overlapping tumor types with IBC renders the differentiation difficult.

Sry-related high-mobility-group/HMG box 10 (SOX10) as a sensitive marker for triple-negative breast cancer.

AIMS: Confirmation of a breast origin for triple-negative breast cancer (TNBC) is sometimes problematic. The traditional breast markers GATA-binding protein 3 (GATA3), mammaglobin (MGB) and gross cystic disease fluid protein 15 (GCDFP15) have shown limitations in identifying TNBC. Here, we aimed to examine the diagnostic potential of the newly proposed TNBC marker, Sry-related high-mobility-group/HMG box 10 (SOX10). METHODS AND RESULTS: We analysed and compared SOX10 expression with GATA3, MGB and GCDFP15 expression in a test cohort of 1838 invasive breast cancers (IBCs) by using tissue microarrays. The findings from the test cohort were further examined with a validation cohort of 42 TNBCs in whole sections. The overall expression rates of SOX10, GATA3, MGB and GCDFP15 were 6.9%, 83.1%, 47.0%, and 34.8%, respectively. Among the TNBCs within this cohort, the expression rates of SOX10, GATA3, MGB and GCDFP15 were 31.3%, 34.5%, 27.9%, and 25.2%, respectively. SOX10 was strongly associated with TNBC (P < 0.001), whereas all other traditional markers were associated with non-TNBC (P < 0.001 for all). In addition, SOX10 was more correlated to basal-like breast cancer (BLBC) (P = 0.001) than five-marker-negative subtype among the TNBCs. A high expression rate of SOX10 (81%) was confirmed in the validation cohort. Additionally, SOX10 expression was inversely correlated with GATA3 and GCDFP15 expression, so they may complement each other in TNBC detection. The SOX10-GATA3 combination yielded a sensitivity of 60.3% for TNBC detection in the test cohort. CONCLUSION: SOX10 is a reliable marker for identifying TNBC, and complements GATA3. The SOX10-GATA3 combination may be used as a sensitive TNBC marker.

Pannexin 1 Channels as an Unexpected New Target of the Anti-Hypertensive Drug Spironolactone.

RATIONALE: Resistant hypertension is a major health concern with unknown cause. Spironolactone is an effective antihypertensive drug, especially for patients with resistant hypertension, and is considered by the World Health Organization as an essential medication. Although spironolactone can act at the mineralocorticoid receptor (MR; NR3C2), there is increasing evidence of MR-independent effects of spironolactone. OBJECTIVE: Here, we detail the unexpected discovery that Panx1 (pannexin 1) channels could be a relevant in vivo target of spironolactone. METHODS AND RESULTS: First, we identified spironolactone as a potent inhibitor of Panx1 in an unbiased small molecule screen, which was confirmed by electrophysiological analysis. Next, spironolactone inhibited α-adrenergic vasoconstriction in arterioles from mice and hypertensive humans, an effect dependent on smooth muscle Panx1, but independent of the MR NR3C2. Last, spironolactone acutely lowered blood pressure, which was dependent on smooth muscle cell expression of Panx1 and independent of NR3C2. This effect, however, was restricted to steroidal MR antagonists as a nonsteroidal MR antagonist failed to reduced blood pressure. CONCLUSIONS: These data suggest new therapeutic modalities for resistant hypertension based on Panx1 inhibition.

Unexpected link between an antibiotic, pannexin channels and apoptosis.

Plasma membrane pannexin 1 channels (PANX1) release nucleotide find-me signals from apoptotic cells to attract phagocytes. Here we show that the quinolone antibiotic trovafloxacin is a novel PANX1 inhibitor, by using a small-molecule screen. Although quinolones are widely used to treat bacterial infections, some quinolones have unexplained side effects, including deaths among children. PANX1 is a direct target of trovafloxacin at drug concentrations seen in human plasma, and its inhibition led to dysregulated fragmentation of apoptotic cells. Genetic loss of PANX1 phenocopied trovafloxacin effects, revealing a non-redundant role for pannexin channels in regulating cellular disassembly during apoptosis. Increase in drug-resistant bacteria worldwide and the dearth of new antibiotics is a major human health challenge. Comparing different quinolone antibiotics suggests that certain structural features may contribute to PANX1 blockade. These data identify a novel linkage between an antibiotic, pannexin channels and cellular integrity, and suggest that re-engineering certain quinolones might help develop newer antibacterials.