Identification of two anti-Candida antibodies associated with the survival of patients with candidemia.

IMPORTANCE: Candidemia (bloodstream invasion by Candida species) is a major fungal disease in humans. Despite the recent progress in diagnosis and treatment, therapeutic options are limited and under threat of antimicrobial resistance. The disease mortality remains high (around 40%). In contrast with deep-seated invasive candidiasis, particularly that occurring in patients with hematologic malignancies and organ transplants, patients with candidemia are often not immunocompromised and therefore able to mount memory anticandidal immune responses, perhaps primed by Candida commensalism. We investigated antibody immunity in candidemia patients and report here on the ability of these patients to produce antibodies that react with Candida antigens. In particular, the patients with high titers of IgG reactive with two immunodominant, virulence-associated antigens (Als3 and MP65) had a higher 30-day survival. If confirmed by controlled, prospective clinical studies, our data could inform the development of antibody therapy to better treat a severe fungal infection such as candidiasis.

Pyrvinium Pamoate Induces Death of Triple-Negative Breast Cancer Stem-Like Cells and Reduces Metastases through Effects on Lipid Anabolism.

Cancer stem-like cells (CSC) induce aggressive tumor phenotypes such as metastasis formation, which is associated with poor prognosis in triple-negative breast cancer (TNBC). Repurposing of FDA-approved drugs that can eradicate the CSC subcompartment in primary tumors may prevent metastatic disease, thus representing an effective strategy to improve the prognosis of TNBC. Here, we investigated spheroid-forming cells in a metastatic TNBC model. This strategy enabled us to specifically study a population of long-lived tumor cells enriched in CSCs, which show stem-like characteristics and induce metastases. To repurpose FDA-approved drugs potentially toxic for CSCs, we focused on pyrvinium pamoate (PP), an anthelmintic drug with documented anticancer activity in preclinical models. PP induced cytotoxic effects in CSCs and prevented metastasis formation. Mechanistically, the cell killing effects of PP were a result of inhibition of lipid anabolism and, more specifically, the impairment of anabolic flux from glucose to cholesterol and fatty acids. CSCs were strongly dependent upon activation of lipid biosynthetic pathways; activation of these pathways exhibited an unfavorable prognostic value in a cohort of breast cancer patients, where it predicted high probability of metastatic dissemination and tumor relapse. Overall, this work describes a new approach to target aggressive CSCs that may substantially improve clinical outcomes for patients with TNBC, who currently lack effective targeted therapeutic options. SIGNIFICANCE: These findings provide preclinical evidence that a drug repurposing approach to prevent metastatic disease in TNBC exploits lipid anabolism as a metabolic vulnerability against CSCs in primary tumors.

Differential Redox State Contributes to Sex Disparities in the Response to Influenza Virus Infection in Male and Female Mice.

Influenza virus replicates intracellularly exploiting several pathways involved in the regulation of host responses. The outcome and the severity of the infection are thus strongly conditioned by multiple host factors, including age, sex, metabolic, and redox conditions of the target cells. Hormones are also important determinants of host immune responses to influenza and are recently proposed in the prophylaxis and treatment. This study shows that female mice are less susceptible than males to mouse-adapted influenza virus (A/PR8/H1N1). Compared with males, PR8-infected females display higher survival rate (+36%), milder clinical disease, and less weight loss. They also have milder histopathological signs, especially free alveolar area is higher than that in males, even if pro-inflammatory cytokine production shows slight differences between sexes; hormone levels, moreover, do not vary significantly with infection in our model. Importantly, viral loads (both in terms of viral M1 RNA copies and tissue culture infectious dose 50%) are lower in PR8-infected females. An analysis of the mechanisms contributing to sex disparities observed during infection reveals that the female animals have higher total antioxidant power in serum and their lungs are characterized by increase in (i) the content and biosynthesis of glutathione, (ii) the expression and activity of antioxidant enzymes (peroxiredoxin 1, catalase, and glutathione peroxidase), and (iii) the expression of the anti-apoptotic protein Bcl-2. By contrast, infected males are characterized by high expression of NADPH oxidase 4 oxidase and phosphorylation of p38 MAPK, both enzymes promoting viral replication. All these factors are critical for cell homeostasis and susceptibility to infection. Reappraisal of the importance of the host cell redox state and sex-related effects may be useful in the attempt to develop more tailored therapeutic interventions in the fight against influenza.

Disruption of IFN-I Signaling Promotes HER2/Neu Tumor Progression and Breast Cancer Stem Cells.

Type I interferon (IFN-I) is a class of antiviral immunomodulatory cytokines involved in many stages of tumor initiation and progression. IFN-I acts directly on tumor cells to inhibit cell growth and indirectly by activating immune cells to mount antitumor responses. To understand the role of endogenous IFN-I in spontaneous, oncogene-driven carcinogenesis, we characterized tumors arising in HER2/neu transgenic (neuT) mice carrying a nonfunctional mutation in the IFNI receptor (IFNAR1). Such mice are unresponsive to this family of cytokines. Compared with parental neu+/- mice (neuT mice), IFNAR1-/- neu+/- mice (IFNAR-neuT mice) showed earlier onset and increased tumor multiplicity with marked vascularization. IFNAR-neuT tumors exhibited deregulation of genes having adverse prognostic value in breast cancer patients, including the breast cancer stem cell (BCSC) marker aldehyde dehydrogenase-1A1 (ALDH1A1). An increased number of BCSCs were observed in IFNAR-neuT tumors, as assessed by ALDH1A1 enzymatic activity, clonogenic assay, and tumorigenic capacity. In vitro exposure of neuT+ mammospheres and cell lines to antibodies to IFN-I resulted in increased frequency of ALDH+ cells, suggesting that IFN-I controls stemness in tumor cells. Altogether, these results reveal a role of IFN-I in neuT-driven spontaneous carcinogenesis through intrinsic control of BCSCs. Cancer Immunol Res; 6(6); 658-70. ©2018 AACR.

Computational drugs repositioning identifies inhibitors of oncogenic PI3K/AKT/P70S6K-dependent pathways among FDA-approved compounds.

The discovery of inhibitors for oncogenic signalling pathways remains a key focus in modern oncology, based on personalized and targeted therapeutics. Computational drug repurposing via the analysis of FDA-approved drug network is becoming a very effective approach to identify therapeutic opportunities in cancer and other human diseases. Given that gene expression signatures can be associated with specific oncogenic mutations, we tested whether a "reverse" oncogene-specific signature might assist in the computational repositioning of inhibitors of oncogenic pathways. As a proof of principle, we focused on oncogenic PI3K-dependent signalling, a molecular pathway frequently driving cancer progression as well as raising resistance to anticancer-targeted therapies. We show that implementation of "reverse" oncogenic PI3K-dependent transcriptional signatures combined with interrogation of drug networks identified inhibitors of PI3K-dependent signalling among FDA-approved compounds. This led to repositioning of Niclosamide (Niclo) and Pyrvinium Pamoate (PP), two anthelmintic drugs, as inhibitors of oncogenic PI3K-dependent signalling. Niclo inhibited phosphorylation of P70S6K, while PP inhibited phosphorylation of AKT and P70S6K, which are downstream targets of PI3K. Anthelmintics inhibited oncogenic PI3K-dependent gene expression and showed a cytostatic effect in vitro and in mouse mammary gland. Lastly, PP inhibited the growth of breast cancer cells harbouring PI3K mutations. Our data indicate that drug repositioning by network analysis of oncogene-specific transcriptional signatures is an efficient strategy for identifying oncogenic pathway inhibitors among FDA-approved compounds. We propose that PP and Niclo should be further investigated as potential therapeutics for the treatment of tumors or diseases carrying the constitutive activation of the PI3K/P70S6K signalling axis.

DNA Damage and Repair Biomarkers in Cervical Cancer Patients Treated with Neoadjuvant Chemotherapy: An Exploratory Analysis.

Cervical cancer cells commonly harbour a defective G1/S checkpoint owing to the interaction of viral oncoproteins with p53 and retinoblastoma protein. The activation of the G2/M checkpoint may thus become essential for protecting cancer cells from genotoxic insults, such as chemotherapy. In 52 cervical cancer patients treated with neoadjuvant chemotherapy, we investigated whether the levels of phosphorylated Wee1 (pWee1), a key G2/M checkpoint kinase, and γ-H2AX, a marker of DNA double-strand breaks, discriminated between patients with a pathological complete response (pCR) and those with residual disease. We also tested the association between pWee1 and phosphorylated Chk1 (pChk1), a kinase acting upstream Wee1 in the G2/M checkpoint pathway. pWee1, γ-H2AX and pChk1 were retrospectively assessed in diagnostic biopsies by immunohistochemistry. The degrees of pWee1 and pChk1 expression were defined using three different classification methods, i.e., staining intensity, Allred score, and a multiplicative score. γ-H2AX was analyzed both as continuous and categorical variable. Irrespective of the classification used, elevated levels of pWee1 and γ-H2AX were significantly associated with a lower rate of pCR. In univariate and multivariate analyses, pWee1 and γ-H2AX were both associated with reduced pCR. Internal validation conducted through a re-sampling without replacement procedure confirmed the robustness of the multivariate model. Finally, we found a significant association between pWee1 and pChk1. The message conveyed by the present analysis is that biomarkers of DNA damage and repair may predict the efficacy of neoadjuvant chemotherapy in cervical cancer. Further studies are warranted to prospectively validate these encouraging findings.

Predictive significance of DNA damage and repair biomarkers in triple-negative breast cancer patients treated with neoadjuvant chemotherapy: An exploratory analysis.

Response of cancer cells to chemotherapy-induced DNA damage is regulated by the ATM-Chk2 and ATR-Chk1 pathways. We investigated the association between phosphorylated H2AX (γ-H2AX), a marker of DNA double-strand breaks that trigger the ATM-Chk2 cascade, and phosphorylated Chk1 (pChk1), with pathological complete response (pCR) in triple-negative breast cancer (TNBC) patients treated with neoadjuvant chemotherapy. γ-H2AX and pChk1 were retrospectively assessed by immunohistochemistry in a series of pretreatment biopsies related to 66 patients. In fifty-three tumors hormone receptor status was negative in both the diagnostic biopsies and residual cancers, whereas in 13 cases there was a slight hormone receptor expression that changed after chemotherapy. Internal validation was carried out. In the entire cohort elevated levels of γ-H2AX, but not pChk1, were associated with reduced pCR rate (p = 0.009). The association tested significant in both uni- and multivariate logistic regression models (OR 4.51, 95% CI: 1.39-14.66, p = 0.012, and OR 5.07, 95% CI: 1.28-20.09, p = 0.021, respectively). Internal validation supported the predictive value of the model. The predictive ability of γ-H2AX was further confirmed in the multivariate model after exclusion of tumors that underwent changes in hormone receptor status during chemotherapy (OR 7.07, 95% CI: 1.39-36.02, p = 0.018). Finally, in residual diseases a significant decrease of γ-H2AX levels was observed (p < 0.001). Overall, γ-H2AX showed ability to predict pCR in TNBC and deserves larger, prospective studies.

The Hippo transducers TAZ and YAP in breast cancer: oncogenic activities and clinical implications.

The Hippo signalling is emerging as a tumour suppressor pathway whose function is regulated by an intricate network of intracellular and extracellular cues. Defects in the signal cascade lead to the activation of the Hippo transducers TAZ and YAP. Compelling preclinical evidence showed that TAZ/YAP are often aberrantly engaged in breast cancer (BC), where their hyperactivation culminates into a variety of tumour-promoting functions such as epithelial-to-mesenchymal transition, cancer stem cell generation and therapeutic resistance. Having acquired a more thorough understanding in the biology of TAZ/YAP, and the molecular outputs they elicit, has prompted a first wave of exploratory, clinically-focused analyses aimed at providing initial hints on the prognostic/predictive significance of their expression. In this review, we discuss oncogenic activities linked with TAZ/YAP in BC, and we propose clinical strategies for investigating their role as biomarkers in the clinical setting. Finally, we address the therapeutic potential of TAZ/YAP targeting and the modalities that, in our opinion, should be pursued in order to further study the biological and clinical consequences of their inhibition.

The Hippo transducer TAZ as a biomarker of pathological complete response in HER2-positive breast cancer patients treated with trastuzumab-based neoadjuvant therapy.

Activation of the Hippo transducer TAZ is emerging as a novel oncogenic route in breast cancer and it has been associated with breast cancer stem cells. Additionally, TAZ expression has been linked with HER-2 positivity. We investigated the association between TAZ expression and pathological complete response in HER2-positive breast cancer patients treated with trastuzumab-based neoadjuvant therapy.TAZ was assessed in diagnostic core biopsies by immunohistochemistry. To categorize samples with low TAZ and samples with high TAZ we generated a score by combining staining intensity and cellular localization. The pathological complete response rate was 78.6% in patients with low TAZ tumors and 57.6% in patients with high TAZ tumors (p=0.082). In HER2-enriched tumors there was no significant association between TAZ and pathological complete response, whereas in the luminal B subtype the pathological complete response rate was 82.4% in tumors with low TAZ and 44.4% in tumors with high TAZ (p=0.035). This association remained statistically significant when restricting our analysis to triple-positive tumors with expression of both estrogen receptor and progesterone receptor ≥ 50% (p=0.035). Results from this exploratory study suggest that the TAZ score efficiently predicts pathological complete response in Luminal B, HER2-positive breast cancer patients who received neoadjuvant chemotherapy and trastuzumab.

Erythropoietin activates cell survival pathways in breast cancer stem-like cells to protect them from chemotherapy.

Recombinant erythropoietin (EPO) analogs [erythropoiesis-stimulating agents (ESA)] are clinically used to treat anemia in patients with cancer receiving chemotherapy. After clinical trials reporting increased adverse events and/or reduced survival in ESA-treated patients, concerns have been raised about the potential role of ESAs in promoting tumor progression, possibly through tumor cell stimulation. However, evidence is lacking on the ability of EPO to directly affect cancer stem-like cells, which are thought to be responsible for tumor progression and relapse. We found that breast cancer stem-like cells (BCSC) isolated from patient tumors express the EPO receptor and respond to EPO treatment with increased proliferation and self-renewal. Importantly, EPO stimulation increased BCSC resistance to chemotherapeutic agents and activated cellular pathways responsible for survival and drug resistance. Specifically, the Akt and ERK pathways were activated in BCSC at early time points following EPO treatment, whereas Bcl-xL levels increased at later times. In vivo, EPO administration counteracted the effects of chemotherapeutic agents on BCSC-derived orthotopic tumor xenografts and promoted metastatic progression both in the presence and in the absence of chemotherapy treatment. Altogether, these results indicate that EPO acts directly on BCSC by activating specific survival pathways, resulting in BCSC protection from chemotherapy and enhanced tumor progression.

Prevention of chemotherapy-induced anemia and thrombocytopenia by constant administration of stem cell factor.

PURPOSE: Chemotherapy-induced apoptosis of immature hematopoietic cells is a major cause of anemia and thrombocytopenia in cancer patients. Although hematopoietic growth factors such as erythropoietin and colony-stimulating factors cannot prevent the occurrence of drug-induced myelosuppression, stem cell factor (SCF) has been previously shown to protect immature erythroid and megakaryocytic cells in vitro from drug-induced apoptosis. However, the effect of SCF in vivo as a single myeloprotective agent has never been elucidated. EXPERIMENTAL DESIGN: The ability of SCF to prevent the occurrence of chemotherapy-induced anemia and thrombocytopenia was tested in a mouse model of cisplatin-induced myelosuppression. To highlight the importance of maintaining a continuous antiapoptotic signal in immature hematopoietic cells, we compared two treatment schedules: in the first schedule, SCF administration was interrupted during chemotherapy treatment and resumed thereafter, whereas in the second schedule, SCF was administered without interruption for 7 days, including the day of chemotherapy treatment. RESULTS: The administration of SCF to cisplatin-treated mice could preserve bone marrow integrity, inhibit apoptosis of erythroid and megakaryocytic precursors, prevent chemotherapy-induced anemia, and rapidly restore normal platelet production. Treatment with SCF increased the frequency of Bcl-2/Bcl-XL-positive bone marrow erythroid cells and sustained Akt activation in megakaryocytes. Myeloprotection was observed only when SCF was administered concomitantly with cisplatin and kept constantly present during the days following chemotherapy treatment. CONCLUSIONS: SCF treatment can prevent the occurrence of chemotherapy-induced anemia and thrombocytopenia in mice, indicating a potential use of this cytokine in the supportive therapy of cancer patients.

Obesity hormone leptin induces growth and interferes with the cytotoxic effects of 5-fluorouracil in colorectal tumor stem cells.

The incidence of colon cancer has increased in developed countries, possibly due to sedentary lifestyle and high caloric diet. Experimental and epidemiological evidence suggests a link between colon cancer development and adipose tissue-derived circulating hormones. Leptin, a pluripotent cytokine secreted by adipocytes, is a key regulator of appetite and energy balance acting in the brain. On the other hand, leptin also controls many physiological and pathological processes in peripheral organs. Recent studies in colon cancer cell lines and human tumors suggested that leptin and its receptor (ObR) are implicated in colon carcinogenesis, and may serve as new biomarkers and pharmacological targets. Here, we explored, for the first time, whether leptin can affect the biology of colorectal tumor stem cells (CTSCs). We found that our previously established and characterized CTSC clones express ObR and respond to leptin with cell proliferation, activation of the extracellular signal-related kinase (ERK)1/2 and AKT signaling pathways, enhanced growth in soft agar, and improved sphere formation associated with E-cadherin overexpression. Moreover, leptin counteracted cytotoxic effects of 5-fluorouracil, a common colon cancer therapeutic agent. These results suggest that obesity and increased leptin levels might promote colorectal cancer by increasing growth and survival of CTSCs.