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Abstract Clinical cancer imaging focuses on tumor growth rather than metastatic phenotypes. The microtubule-depolymerizing drug, Vinorelbine, reduced the metastatic phenotypes of microtentacles, reattachment and tumor cell clustering more than tumor cell viability. Treating mice with Vinorelbine for only 24 h had no significant effect on primary tumor survival, but median metastatic tumor survival was extended from 8 to 30 weeks. Microtentacle inhibition by Vinorelbine was also detectable within 1 h, using tumor cells isolated from blood samples. As few as 11 tumor cells were sufficient to yield 90% power to detect this 1 h Vinorelbine drug response, demonstrating feasibility with the small number of tumor cells available from patient biopsies. This study establishes a proof-of-concept that targeted microtubule disruption can selectively inhibit metastasis and reveals that existing FDA-approved therapies could have anti-metastatic actions that are currently overlooked when focusing exclusively on tumor growth.

Levels of hydrogen peroxide are highly elevated in the breast tumor microenvironment compared to normal tissue. Production of hydrogen peroxide is implicated in the mechanism of action of many anticancer therapies. Several lines of evidence suggest hydrogen peroxide mediates breast carcinogenesis and metastasis, though the molecular mechanism remains poorly understood. This study elucidates the effects of exposure to elevated hydrogen peroxide on non-tumorigenic MCF10A mammary epithelial cells, tumorigenic MCF7 cells, and metastatic MDA-MB-231 breast cancer cells. Hydrogen peroxide treatment resulted in a dose- and time-dependent induction of two α-tubulin post-translational modifications—de-tyrosination and acetylation—both of which are markers of poor patient prognosis in breast cancer. Hydrogen peroxide induced the formation of tubulin-based microtentacles in MCF10A and MDA-MB-231 cells, which were enriched in detyrosinated and acetylated α-tubulin. However, the hydrogen peroxide-induced microtentacles did not functionally promote metastatic phenotypes of cellular reattachment and homotypic cell clustering. These data establish for the first time that microtentacle formation can be separated from the functions to promote reattachment and clustering, which indicates that there are functional steps that remain to be identified. Moreover, signals in the primary tumor microenvironment may modulate α-tubulin post-translational modifications and induce microtentacles; however, the functional consequences appear to be context-dependent.

Background: Anal precancers and cancers can be detected during screening with high-resolution anoscopy (HRA). The sensitivity of HRA depends on the burden and duration of human papillomavirus (HPV) among those screened as well as anoscopist proficiency, which is highly correlated with prior screening experience. Our objective was to compare the identification and type of HPV and the likelihood of HRA-detected precancer for men who have sex with men (MSM) undergoing their first HRA-screening in Nigeria. Methods: MSM were recruited from an HIV test-and-treat cohort, TRUST/RV368, into a new anal cancer screening program. Anal swabs obtained during screening underwent Ion Torrent next-generation sequencing using barcoded HPV PCR broad-spectrum primers 5+/6+ to detect up to 161 HPVs. All high-risk (HR) HPVs and the most abundant low-risk (LR)-HPVs were evaluated as type-specific infections with some categorized as belonging to a multiple infection. HRA screening results included benign, low-grade squamous intraepithelial lesions (LSIL), or HSIL as detected by cytology or histology. Multivariable logistic regression was used to assess the association of HPV and other cofactors with any SIL. Results: Among 342 MSM, 60% were HIV-infected, 89% were under 35 years of age, and 51% had 8 or more years since anal coital debut. Of those with SIL, 89% had LSIL and only 11% had HSIL. Prevalence of any HPV and high-risk (HR)-HPV was 92% and 74%, respectively. The most prevalent genotypes in rank order were HPV6 (31%), HPV16 (23%), HPV42 (20%), HPV11 (18%), HPV45 (18%), and HPV51 (17%). For multiple HR-HPVs, 31% had a single HR-HPV, 32% had 2-3, and 10% had 4 or more. Low-risk HPVs, type 6 and/or 11, were common (42%) and were significantly associated with SIL (adjusted odds ratio [aOR]:1.8, 95% confidence interval [CI]: 1.1–3.1) together with perianal warts (aOR:6.7, 95% CI: 3.3–13.5). In contrast, HR-HPV and multiple HR-HPVs were not significantly associated with SIL (all p > 0.05). Conclusions: Detection of HSIL was low. Although HR-HPV was abundant, HSIL development also depends on the duration of HR-HPV infections and the anoscopist's level of experience. As our cohort ages and the anoscopist becomes more skilled, detection of HSIL will likely improve.

Abstract Background Our next generation sequencing (NGS)-based human papillomavirus (HPV) genotyping assay showed a high degree of concordance with the Roche Linear Array (LA) with as little as 1.25 ng formalin-fixed paraffin-embedded-derived genomic DNA in head and neck and cervical cancer samples. This sensitive genotyping assay uses barcoded HPV PCR broad-spectrum general primers 5+/6+ (BSGP)5+/6+ applicable to population studies, but it’s diagnostic performance has not been tested in cases with multiple concurrent HPV infections. Methods We conducted a cross-sectional study to compare the positive and negative predictive value (PPV and NPV), sensitivity and specificity of the NGS assay to detect HPV genotype infections as compared to the LA. DNA was previously extracted from ten anal swab samples from men who have sex with men in Nigeria enrolled on the TRUST/RV368 cohort study. Two-sample tests of proportions were used to examine differences in the diagnostic performance of the NGS assay to detect high vs. low-risk HPV type-specific infections. Results In total there were 94 type-specific infections detected in 10 samples with a median of 9.5, range (9 to 10) per sample. Using the LA as the gold standard, 84.4% (95% CI: 75.2–91.2) of the same anal type-specific infections detected on the NGS assay had been detected by LA. The PPV and sensitivity differed significantly for high risk (PPV: 90%, 95% CI: 79.5–96.2; sensitivity: 93.1%, 95% CI: 83.3–98.1) as compared to low risk HPV (PPV: 73%, 95% CI: 54.1–87.7; sensitivity: 61.1, 95% CI: 43.5–76.9) (all p 0.05). The NGS assay detected 10 HPV genotypes that were not among the 37 genotypes found on LA (30, 32, 43, 44, 74, 86, 87, 90, 91, 114). Conclusions The NGS assay accurately detects multiple HPV infections in individual clinical specimens with limited sample volume and has extended coverage compared to LA.

Long noncoding RNA differentiation antagonizing nonprotein coding RNA (lncRNA-DANCR) is associated with poor prognosis in multiple cancers, and promotes cancer stemness and invasion. However, the exact mechanisms by which DANCR promotes non-small cell lung cancer (NSCLC) remain elusive. In this study, we determined that DANCR knockdown (KD) impeded cell migration and reduced stem-like characteristics in two NSCLC cell lines, A549 and H1755. Wnt signaling was shown to promote NSCLC proliferation, stemness, and invasion; therefore, we hypothesized that DANCR may regulate these activities through induction of the Wnt/β-catenin pathway. DANCR KD reduced β-catenin signaling and protein expression, and decreased the expression of β-catenin gene targets c-Myc and Axin2. One of the well-defined functions of lncRNAs is their ability to bind and inhibit microRNAs. Through in silico analysis, we identified tumor suppressor miR-216a as a potential binding partner to DANCR, and confirmed this binding through coimmunoprecipitation and luciferase-reporter assays. Furthermore, we show that DANCR-induced β-catenin protein expression may be blocked with miR-216a overexpression. Our findings illustrate a role of DANCR in NSCLC migration and stemness, and suggest a novel DANCR/miR-216a signaling axis in the Wnt/β-catenin pathway.

The oral second-generation bis-aryl urea fms-like tyrosine kinase 3 (FLT3) inhibitor quizartinib (AC220) has favorable kinase selectivity and pharmacokinetics. It inhibits mutant and wild-type FLT3 in vivo at 0.1 and 0.5 µM, respectively, and has shown favorable activity and tolerability in phase I and II trials in acute myeloid leukemia, with QT prolongation as the dose-limiting toxicity. Co-administration with chemotherapy is planned. We characterized interactions of quizartinib with the ATP-binding cassette (ABC) proteins ABCB1 (P-glycoprotein) and ABCG2 (breast cancer resistance protein). Its effects on uptake of fluorescent substrates and apoptosis were measured by flow cytometry, binding to ABCB1 and ABCG2 drug-binding sites by effects on [¹²⁵I]iodoarylazidoprazosin ([¹²⁵I]-IAAP) photolabeling and ATPase activity, and cell viability by the WST-1 colorimetric assay. Quizartinib inhibited transport of fluorescent ABCG2 and ABCB1 substrates in ABCG2- and ABCB1-overexpressing cells in a concentration-dependent manner, from 0.1 to 5 µM and from 0.5 to 10 µM, respectively, and inhibited [¹²⁵I]-IAAP photolabeling of ABCG2 and ABCB1 with IC₅₀ values of 0.07 and 3.3 µM, respectively. Quizartinib at higher concentrations decreased ABCG2, but not ABCB1, ATPase activity. Co-incubation with quizartinib at 0.1 to 1 µM sensitized ABCG2-overexpressing K562/ABCG2 and 8226/MR20 cells to ABCG2 substrate chemotherapy drugs in a concentration-dependent manner in cell viability and apoptosis assays. Additionally, quizartinib increased cellular uptake of the ABCG2 substrate fluoroquinolone antibiotic ciprofloxacin, which also prolongs the QT interval, in a concentration-dependent manner, predicting altered ciprofloxacin pharmacokinetics and pharmacodynamics when co-administered with quizartinib. Thus quizartinib inhibits ABCG2 at pharmacologically relevant concentrations, with implications for both chemosensitization and adverse drug interactions. These interactions should be considered in the design of treatment regimens combining quizartinib and chemotherapy drugs and in choice of concomitant medications to be administered with quizartinib.