ROR2 Promotes EMT and Necrosis by Hyperactivating ERK in Melanoma

PurposeReceptor tyrosine kinase-like orphan receptor 2 (ROR2) has been shown to play opposite roles in the progression of different tumor types. In melanoma, ROR2 was shown to associate with a more invasive phenotype and to contribute to experimental lung colonization. However, the underlying mechanisms regulated by ROR2 have not been elucidated. More importantly, it has not been established whether ROR2 is just a marker or a driver of melanoma aggressiveness. MethodsGain and loss-of-function experiments were applied to study the biological function of ROR2 in melanoma. Transwell assays and Western blots were used to evaluate cell migration and both expression and activation of Epithelial-Mesenchymal Transition (EMT) markers and signaling proteins. The role of ROR2 in vivo was assessed in xenotransplantation experiments.ResultsWe describe that ROR2 promotes EMT by inducing cadherin switch and the upregulation of the transcription factors ZEB1, Twist, Slug, Snail, and HIF1A, together with a mesenchymal phenotype and increased migration. ROR2 association with EMT is also observed in melanoma samples. ROR2 exerts these effects by hyperactivating the MAPK/ERK pathway far above the typical high constitutive activity observed in melanoma. ROR2 also promoted EMT, invasion, and necrosis in xenotransplanted mice. This important role of ROR2 translates into melanoma patient’s prognosis since patients with lymph node metastasis displaying elevated ROR2 levels have reduced overall survival and distant metastasis-free survival.Conclusions These results demonstrate that ROR2 contributes to melanoma progression by hyperactivating ERK and inducing EMT and necrosis. Thus, ROR2 can be an attractive therapeutic target for metastatic melanoma.

Interferon-Gamma Signaling Promotes Melanoma Progression and Metastasis

Interferon-gamma (IFNG) has long been regarded as the flag-bearer for the anti-cancer immunosurveillance mechanisms. However, relatively recent studies have suggested a dual role of IFNG, albeit there is no direct experimental evidence for its potential pro-tumor functions. Here we provide in vivo evidence that treatment of mouse melanoma cell lines with physiological levels of Ifng enhances their tumorigenicity and metastasis in lung colonization allograft assays performed in immunocompetent syngeneic host mice, but not in immunocompromised host mice. We also show that this enhancement is dependent on downstream signaling via Stat1 but not Stat3, providing evidence of an oncogenic function of Stat1 in melanoma. The experimental results suggest that melanoma cell-specific Ifng signaling modulates the tumor microenvironment and its pro-tumorigenic effects are dependent on the γδ T cells, as Ifng-enhanced tumorigenesis was inhibited in the TCR-δ knockout mice. Overall, these results show that Ifng signaling may have tumor-promoting effects in melanoma by modulating the immune cell composition of the tumor microenvironment.

Outcomes of nontuberculous mycobacteria isolation among lung transplant recipients: A matched case-control with retrospective cohort study

Disclaimer In an effort to expedite the publication of articles , AJHP is posting manuscripts online as soon as possible after acceptance. Accepted manuscripts have been peer-reviewed and copyedited, but are posted online before technical formatting and author proofing. These manuscripts are not the final version of record and will be replaced with the final article (formatted per AJHP style and proofed by the authors) at a later time. Purpose Lung transplant recipients are at increased risk for acquiring nontuberculous mycobacteria (NTM), but the clinical significance of NTM isolation, particularly among patients not meeting guideline-endorsed diagnostic criteria for NTM pulmonary disease, is unclear. Methods A case-control study of lung transplant recipients with culture-positive NTM infections treated at a large transplant center during a 7-year period (2013-2019) was performed. Results Twenty-nine cases were matched 1:2 to non-NTM controls. The median time to NTM isolation was 10.7 months post transplant. Only 34.5% of all cases, and half of treated cases, met diagnostic criteria for NTM pulmonary infection. All-cause mortality at 12 months was numerically higher among NTM cases versus controls (20.7% vs 8.6%, P = 0.169); however, no deaths were attributed to NTM. No increase in the 12-month rate of acute rejection was observed (27.6% vs 36.2%, P = 0.477). Recent augmented immunosuppression was associated with increased odds of NTM isolation, while azithromycin prophylaxis was associated with reduced odds of isolation and was not associated with macrolide resistance. Both adverse events and actual or potential drug-drug interactions occurred in more than 90% of treated cases; these events included ocular toxicity, hearing loss, and supratherapeutic calcineurin inhibitor concentrations. Eight of the 14 treated cases (57.1%) required early antibiotic discontinuation due to adverse events or drug-drug interactions. Conclusion Among lung transplant recipients, most patients with NTM isolation did not meet guideline criteria for infection and had outcomes similar to non‒NTM-infected patients, which may reflect transient lung colonization by NTM rather than true disease. As adverse events are common with NTM therapy, limiting unnecessary antibiotic treatment represents an area for future antimicrobial stewardship efforts.

ICAM1 initiates CTC cluster formation and trans-endothelial migration in lung metastasis of breast cancer

Circulating tumor cell (CTC) clusters mediate metastasis at a higher efficiency and are associated with lower overall survival in breast cancer compared to single cells. Combining single-cell RNA sequencing and protein analyses, here we report the profiles of primary tumor cells and lung metastases of triple-negative breast cancer (TNBC). ICAM1 expression increases by 200-fold in the lung metastases of three TNBC patient-derived xenografts (PDXs). Depletion of ICAM1 abrogates lung colonization of TNBC cells by inhibiting homotypic tumor cell-tumor cell cluster formation. Machine learning-based algorithms and mutagenesis analyses identify ICAM1 regions responsible for homophilic ICAM1-ICAM1 interactions, thereby directing homotypic tumor cell clustering, as well as heterotypic tumor-endothelial adhesion for trans-endothelial migration. Moreover, ICAM1 promotes metastasis by activating cellular pathways related to cell cycle and stemness. Finally, blocking ICAM1 interactions significantly inhibits CTC cluster formation, tumor cell transendothelial migration, and lung metastasis. Therefore, ICAM1 can serve as a novel therapeutic target for metastasis initiation of TNBC.

Antineoplastic Activity of Products Derived From Cellulose-containing Materials: Study of Levoglucosenone and Structurally-related Analogous as New Alternatives for Breast Cancer Treatment

Purpose: Breast cancer is the leading cause of cancer death among women worldwide. For this reason, the development of new therapies is still essential. In this work we have analyzed the antitumor potential of levoglucosenone, a chiral building block derived from glucose, and three structurally related analogues obtained from soybean hulls pyrolysis.Methods: Employing human and murine mammary cancer models, we have evaluated the effect of our compounds on cell viability through MTS assay, apoptosis induction by acridine orange / ethidium bromide staining and/or flow cytometry and the loss of mitochondrial potential by tetramethylrhodamine methyl ester staining. Autophagy and senescence induction were also evaluated by Western blot and β-galactosidase activity respectively. Secreted metalloproteases activity was determined by quantitative zymography. Migratory capacity was assessed by wound healing assays while invasive potential was analyzed using Matrigel-coated transwell chambers. In vivo studies were also performed to evaluate subcutaneous tumor growth and experimental lung colonization.Results: Apoptosis was identified as the main mechanism responsible for the reduction of monolayer cell content induced by the compounds without detecting modulations of autophagy or senescence processes. Two of the four compounds were able to modulate in vitro events associated with tumor progression, such as migratory potential, invasiveness, and proteases secretion. Furthermore, tumor volume and metastatic spread were significantly reduced in vivo after treatment with the compounds.Conclusion: We could obtain from soybean hulls, a material with almost no commercial value, a variety of chemical compounds useful for breast cancer treatment.

Long-term coexistence of Pseudomonas aeruginosa and Staphylococcus aureus using an in vitro cystic fibrosis model

Aim: To investigate the role of pre-established Staphylococcus aureus on Pseudomonas aeruginosa adaptation and antibiotic tolerance. Materials & methods: Bacteria were cultured mimicking the sequential pattern of lung colonization and exposure to ciprofloxacin. Results: In the absence of ciprofloxacin exposure, S. aureus and P. aeruginosa coexisted supported by the physicochemical characteristics of the artificial sputum medium. S. aureus had no role in P. aeruginosa tolerance against ciprofloxacin and did not select P. aeruginosa small-colony variants during antibiotic treatment. rhlR and psqE were downregulated after the contact with S. aureus indicating that P. aeruginosa attenuated its virulence potential. Conclusion: P. aeruginosa and S. aureus can cohabit in cystic fibrosis airway environment for long-term without significant impact on P. aeruginosa adaptation and antibiotic tolerance.

Novel role of the dietary flavonoid fisetin in suppressing rRNA biogenesis

The nucleolus of a cell is a critical cellular compartment that is responsible for ribosome biogenesis and plays a central role in tumor progression. Fisetin, a nutraceutical, is a naturally occurring flavonol from the flavonoid group of polyphenols that has anti-cancer effects. Fisetin negatively impacts several signaling pathways that support tumor progression. However, effect of fisetin on the nucleolus and its functions were unknown. We observed that fisetin is able to physically enter the nucleolus. In the nucleolus, RNA polymerase I (RNA Pol I) mediates the biogenesis of ribosomal RNA. Thus, we investigated the impacts of fisetin on the nucleolus. We observed that breast tumor cells treated with fisetin show a 20–30% decreased nucleolar abundance per cell and a 30–60% downregulation of RNA Pol I transcription activity, as well as a 50–70% reduction in nascent rRNA synthesis, depending on the cell line. Our studies show that fisetin negatively influences MAPK/ERK pathway to impair RNA Pol I activity and rRNA biogenesis. Functionally, we demonstrate that fisetin acts synergistically (CI = 0.4) with RNA Pol I inhibitor, BMH-21 and shows a noteworthy negative impact (60% decrease) on lung colonization of breast cancer cells. Overall, our findings highlight the potential of ribosomal RNA (rRNA) biogenesis as a target for secondary prevention and possible treatment of metastatic disease.

Neutrophil-derived reactive oxygen species promote tumor colonization

A single-nucleotide polymorphism of neutrophil cytosolic factor 1 (Ncf1), leading to an impaired generation of reactive oxygen species (ROS), is a causative genetic factor for autoimmune disease. To study a possible tumor protection effect by the Ncf1 mutation in a manner dependent on cell types, we used experimental mouse models of lung colonization assay by B16F10 melanoma cells. We observed fewer tumor foci in Ncf1 mutant mice, irrespective of αβT, γδT, B-cell deficiencies, or of a functional Ncf1 expression in CD68-positive monocytes/macrophages. The susceptibility to tumor colonization was restored by the human S100A8 (MRP8) promoter directing a functional Ncf1 expression to granulocytes. This effect was associated with an increase of both ROS and interleukin 1 beta (IL-1β) production from lung neutrophils. Moreover, neutrophil depletion by anti-Ly6G antibodies increased tumor colonization in wild type but failed in the Ncf1 mutant mice. In conclusion, tumor colonization is counteracted by ROS-activated and IL-1β-secreting tissue neutrophils.

Stress-Inducible Gene Atf3 Dictates a Dichotomous Macrophage Activity in Chemotherapy-Enhanced Lung Colonization

Previously, we showed that chemotherapy paradoxically exacerbated cancer cell colonization at the secondary site in a manner dependent on Atf3, a stress-inducible gene, in the non-cancer host cells. Here, we present evidence that this phenotype is established at an early stage of colonization within days of cancer cell arrival. Using mouse breast cancer models, we showed that, in the wild-type (WT) lung, cyclophosphamide (CTX) increased the ability of the lung to retain cancer cells in the vascular bed. Although CTX did not change the WT lung to affect cancer cell extravasation or proliferation, it changed the lung macrophage to be pro-cancer, protecting cancer cells from death. This, combined with the initial increase in cell retention, resulted in higher lung colonization in CTX-treated than control-treated mice. In the Atf3 knockout (KO) lung, CTX also increased the ability of lung to retain cancer cells. However, the CTX-treated KO macrophage was highly cytotoxic to cancer cells, resulting in no increase in lung colonization—despite the initial increase in cell retention. In summary, the status of Atf3 dictates the dichotomous activity of macrophage: pro-cancer for CTX-treated WT macrophage but anti-cancer for the KO counterpart. This dichotomy provides a mechanistic explanation for CTX to exacerbate lung colonization in the WT but not Atf3 KO lung.