Surgery versus radiotherapy: Long term outcomes of T1 glottic cancer

AimThe aim of this study was to compare the outcomes, patterns of failure and laryngeal preservation rates in patients with T1N0 glottic cancer treated with surgery or radiotherapy.Materials/methodsRetrospective study of T1N0 glottic cancer patients treated in our institution between January 2007 and December 2017. Histologically proven squamous cell carcinoma patients, treated with upfront cordectomy/partial laryngectomy (S group) or radiotherapy (RT group) were included. Elective treatment of the neck was not permitted. Local failure (LF), disease-free survival (DFS), ultimate disease-free survival (UDFS), laryngectomy-free survival (LFS), disease-specific mortality (DSM) and overall survival (OS) were evaluated.ResultsTwo hundred and one patients were eligible (172 S group, 29 RT group), with a median follow-up of 38.8 months. Overall, 33 (16%) patients had a recurrence, 30 (17%) in the S group and 3 (10%) in the RT group. Local failure was the predominant site of failure (28 S, 2 RT). Overall, of all those that were salvaged, 17 (8%) underwent total laryngectomy (15 S, 2 RT). There was no significant difference in the 5-year cumulative incidence of LF (20.8% S, 8.1% RT, p = 0.138), 5-y LFS (85.0% vs. 91.7%, p = 0.809), 5-y DFS (67.5% vs. 82.1%, p = 0.343), 5-y UDFS (82.5% vs. 90.3%, p = 0.647) and 5-y OS (84.5% vs. 90.3%, p = 0.892). Multivariate analysis showed no correlation between initial treatment and the analyzed outcomes.ConclusionPrimary surgery or radiotherapy were similar first line options, since they do not differ in all outcomes. Patients’ and physician's preferences must be considered when choosing first treatment.     

A comparison of a moderately hypofractionated IMRT planning technique used in a randomised UK external beam radiotherapy trial with an in-house technique for localised prostate cancer

AimTo compare the radiotherapy technique used in a randomised trial with VMAT and an in-house technique for prostate cancer.BackgroundTechniques are evolving with volumetric modulated arc therapy (VMAT) commonly used. The CHHiP trial used a 3 PTV forward planned IMRT technique (FP_CH). Our centre has adopted a simpler two PTV technique with locally calculated margins.Materials and methods25 patients treated with FP_CH to 60 Gy in 20 fractions were re-planned with VMAT (VMAT_CH) and a two PTV protocol (VMAT_60/52 and VMAT_60/48). Target coverage, conformity index (CI), homogeneity index (HI), monitor units (MU) and dose to the rectum, bladder, hips and penile bulb were compared.ResultsPTV coverage was high for all techniques. VMAT_CH plans had better CI than FP_CH (p ≤  0.05). VMAT_60/52/48 plans had better CI than VMAT_CH. FP_CH had better HI and fewer MU than VMAT (p ≤  0.05). More favourable rectum doses were found for VMAT _CH than FP_CH (V_48.6, V_52.8, V₅₇, p ≤  0.05) with less difference for bladder (p ≥  0.05). Comparing VMAT_CH to VMAT_60/52/48 showed little differences for the bladder and rectum but VMAT_CH had larger penile bulb doses (V_40.8, V_48.6, mean, D_2, p ≤  0.05). Femoral head doses (V_40.8) were similarly low for all techniques (p = ≥ 0.05).ConclusionVMAT produced more conformal plans with smaller rectum doses compared to FP_CH albeit worse HI and more MU. VMAT_60/52 and VMAT_60/48 plans had similar rectal and bladder doses to VMAT_CH but better CI and penile bulb doses which may reduce toxicity.     

Detection of setup uncertainties with 3D surface registration system for conformal radiotherapy of breast cancer

Aim

To investigate the clinical application of a technique for patient set-up verification in breast cancer radiotherapy based on a 3D surface image registration system.

Background

Accurate and reproducible patient set-up is a prerequisite to correctly deliver fractionated radiotherapy. Various approaches are available to verify and correct patient setup for 3D image acquisition in a radiation treatment room.

Materials and methods

The study analyzed the setup reproducibility of 15 patients affected by breast cancer and candidates for conformal radiotherapy by using the AlignRT system (VisionRT, London, UK). At the initial setup, electronic portal imaging device (EPID) images were compared with Digitally Reconstructed Radiographs (DRRs) and a reference three-dimensional (3D) surface image was obtained by AlignRT. Surface images were acquired prior to every subsequent setup procedure. The systematic and random errors along longitudinal and vertical directions were measured and compared for the two systems.

Results

The procedure for surface registration, image acquisition and comparison with the reference image took less than 1 min on average. The T test for systematic error showed no significant difference between the 2 verification systems along the longitudinal (p = 0.69) and vertical (p = 0.67) axes. The T-test for random error showed a significant difference between the 2 systems along the vertical axis (p = 0.05).

Conclusion

AlignRT is fast, simple, non-invasive and seems to be reliable in detecting patient setup errors. Our results suggest that it could be used to assess the setup reproducibility for breast cancer patients.     

DDX3 acts as a tumor suppressor in colorectal cancer as loss of DDX3 in advanced cancer promotes tumor progression by activating the MAPK pathway

Objective: The treatment and prognosis of patients with advanced colorectal cancer (CRC) remain a difficult problem. Herein, we investigated the role of DEAD (Asp-Glu-Ala-Asp) box helicase 3 (DDX3) in CRC and proposed potential therapeutic targets for advanced CRC.Methods: The expression of DDX3 in CRC and its effect on prognosis were explored by databases and CRC tissue microarrays. Stable DDX3 knockdown and overexpression cell lines were established with lentiviral vectors. The effects of DDX3 on CRC were investigated by functional experiments in vitro and in vivo. The molecular mechanism of DDX3 in CRC was explored by western blotting. Molecular-specific inhibitors were further used to explore potential therapeutic targets for advanced CRC.Results: The expression of DDX3 was decreased in advanced CRC, and patients with low DDX3 expression had a poor prognosis. In vitro and in vivo experiments showed that low DDX3 expression promoted the proliferation, migration and invasion of CRC. DDX3 loss regulated E-cadherin and β-catenin signaling through the mitogen-activated protein kinase (MAPK) pathway as shown by western blotting. In addition, the MEK inhibitor, PD98059, significantly reduced the increased cell proliferation, migration and invasion caused by knockdown of DDX3.Conclusions: DDX3 acts as a tumor suppressor gene in CRC. DDX3 loss in advanced cancer promotes cancer progression by regulating E-cadherin and β-catenin signaling through the MAPK pathway, and targeting the MAPK pathway may be a therapeutic approach for advanced CRC.     

Multifaceted regulation of the sumoylation of the Sgs1 DNA helicase

Homologous recombination repairs DNA breaks and sequence gaps via the production of joint DNA intermediates such as Holliday junctions. Dissolving Holliday junctions into linear DNA repair products requires the activity of the Sgs1 helicase in yeast and of its homologs in other organisms. Recent studies suggest that the functions of these conserved helicases are regulated by sumoylation; however, the mechanisms that promote their sumoylation are not well understood. Here, we employed in vitro sumoylation systems and cellular assays to determine the roles of DNA and the scaffold protein Esc2 in Sgs1 sumoylation. We show that DNA binding enhances Sgs1 sumoylation in vitro. In addition, we demonstrate the Esc2’s midregion (MR) with DNA-binding activity is required for Sgs1 sumoylation. Unexpectedly, we found that the sumoylation-promoting effect of Esc2-MR is DNA independent, suggesting a second function for this domain. In agreement with our biochemical data, we found the Esc2-MR domain, like its SUMO E2-binding C-terminal domain characterized in previous studies, is required for proficient sumoylation of Sgs1 and its cofactors, Top3 and Rmi1, in cells. Taken together, these findings provide evidence that while DNA binding enhances Sgs1 sumoylation, Esc2-based stimulation of this modification is mediated by two distinct domains.     

DNA-DAPI Interaction-Based Method for Cell Proliferation Rate Evaluation in 3D Structures

Effective cell number monitoring throughout the three-dimensional (3D) scaffold is a key factor in tissue engineering. There are many methods developed to evaluate cell number in 2D environments; however, they often encounter limitations in 3D. Therefore, there is a demand for reliable methods to measure cell proliferation in 3D surroundings. Here, we report a novel technique for the DNA content-based evaluation of cell proliferation using DNA-binding dye DAPI. We demonstrated the method’s compatibility with four different cell cultures: cancer lines MCF-7 and MH-22a, embryonic fibroblast cell line Swiss 3T3, and primary mesenchymal stem cell culture isolated from rat’s incisors. The DAPI based method was able to successfully evaluate cell proliferation in 2D, 2.5D, and 3D environments. Even though the proposed method does not discriminate between viable and dead cells, it might give a convenient snapshot of the cell number at a given time point. This should help to more reliably evaluate various processes proceeding in 2.5D and 3D cultures.     

FOXO3a inhibits the EMT and metastasis of breast cancer by regulating TWIST-1 mediated miR-10b/CADM2 axis

BackgroundBreast cancer is the most common malignancy and has been considered as a leading cause of cancer death in women. Exploring the mechanism of breast cancer metastasis is extremely important for seeking novel therapeutic strategies and improving prognosis.MethodsClinical specimens and pathological characteristics were collected for evaluating the expression of forkhead box class O 3a (FOXO3a) and twist-related protein 1 (TWIST-1) in breast cancer tissues. CCK-8 assay was used to analyze cell proliferation. Cell invasion and migration were assessed by transwell assays. The expression of FOXO3a, TWIST-1, miR-10b, CADM2, FAK, phosphor-AKT and the epithelial-mesenchymal transition (EMT)-related protein (N-cadherin, E-cadherin and vimentin) were analyzed by RT-qPCR, immunohistochemical staining, immunofluorescence assay or western blot, respectively. Xenograft mouse models were used to analyze the role of the FOXO3a in breast cancer.ResultsFOXO3a was down-regulated and TWIST-1 was up-regulated in breast cancer tissues. Overexpression of FOXO3a or knockdown of TWIST-1 suppressed the proliferation, invasion, migration and EMT of breast cancer cells. Overexpression of TWIST-1 could reverse the effect of FOXO3a on the proliferation, invasion, migration and EMT of breast cancer. Moreover, FOXO3a suppressed the growth and metastasis of breast cancer by targeting TWIST1 in vivo.ConclusionFOXO3a inhibited the EMT and metastasis of breast cancer via TWIST-1/miR-10b/CADM2 axis.     

Deubiquitinase USP2 stabilizes the MRE11–RAD50–NBS1 complex at DNA double-strand break sites by counteracting the ubiquitination of NBS1

The MRE11–RAD50–NBS1 (MRN) complex plays essential roles in the cellular response to DNA double-strand breaks (DSBs), which are the most cytotoxic DNA lesions, and is a target of various modifications and controls. Recently, lysine 48-linked ubiquitination of NBS1, resulting in premature disassembly of the MRN complex from DSB sites, was observed in cells lacking RECQL4 helicase activity. However, the role and control of this ubiquitination during the DSB response in cells with intact RECQL4 remain unknown. Here, we showed that USP2 counteracts this ubiquitination and stabilizes the MRN complex during the DSB response. By screening deubiquitinases that increase the stability of the MRN complex in RECQL4-deficient cells, USP2 was identified as a new deubiquitinase that acts at DSB sites to counteract NBS1 ubiquitination. We determined that USP2 is recruited to DSB sites in a manner dependent on ATM, a major checkpoint kinase against DSBs, and stably interacts with NBS1 and RECQL4 in immunoprecipitation experiments. Phosphorylation of two critical residues in the N terminus of USP2 by ATM is required for its recruitment to DSBs and its interaction with RECQL4. While inactivation of USP2 alone does not substantially influence the DSB response, we found that inactivation of USP2 and USP28, another deubiquitinase influencing NBS1 ubiquitination, results in premature disassembly of the MRN complex from DSB sites as well as defects in ATM activation and homologous recombination repair abilities. These results suggest that deubiquitinases counteracting NBS1 ubiquitination are essential for the stable maintenance of the MRN complex and proper cellular response to DSBs.     

Heuristic estimation of the α/β ratio for a cohort of Mexican patients with prostate cancer treated with external radiotherapy techniques

BackgroundThe aim of the study was to Estimate and compare the radiobiological ratio α/β with the heuristic method for a cohort of Mexican patients with prostate cancer (PCa) who were treated with external radiotherapy (RT) techniques at three Hospital Institutions in Mexico City. With the Kaplan-Meier technique and the Cox proportional hazards model, the biochemical relapse-free survival (bRFS) is determined and characterized for cohorts of Mexican patients with PCa who received treatment with external RT. Using these clinical outcomes, the radiobiological parameter α/β is determined using the heuristic methodology of Pedicini et. al.Materials and methodsThe α/β is calculated from the survival curves for different treatment schemes implemented at three distinct hospitals. The Pedicini’s techniques allow to determine the parameters α/β, k and N₀ when treatments are not radiobiologically equivalent, therefore, are built up of a set of curved pairs for the biologically effective dose (BED) versus the ratio α/β, where the ratio is given by the intersection for each pair of curves.ResultsSix different values of α/β were found: the first α/β = 2.46 Gy, the second α/β = 3.30 Gy, the third for α/β = 3.25 Gy, the fourth α/β = 3.24 Gy, the fifth α/β = 3.38 Gy and the last α/β = 4.08 Gy. These values can be explained as follows: a) The bRFS of the schemes presents a statistical variation; b) The absorbed doses given to the patient present uncertainties on the physical dosimetry that are not on the modeling; c) Finally, in the model for the bRFS of Eq. (3), there are parameters that have to be considered, such as: the number of clonogenic tumor cells N₀, the overall treatment time (OTT), the kick-off time for tumor repopulation T_k and the repopulation doubling time. Therefore, the mean value to α/β for all schemes has an average value of 3.29 (± 0.52) Gy.ConclusionsThe value of α/β¯=3.29 (±0.52) Gy is determined from cohorts of Mexican patients with PC a treated with external radiotherapy using the time-dependent LQ model, which is a higher value with respect to the “dogma” value of α/β 1.5 Gy obtained with the LQ model without temporal dependence. Therefore, there is a possibility of optimizing treatments radiobiologically and improving the results of bRFS in Mexican patients with PCa treated with external radiotherapy.     

IDKK1 inhibits canonical Wnt signaling in human papillomavirus-positive penile cancer cells

Penile squamous cell cancer (PSCC) is the most frequent penile malignant disease. Infections with human papillomaviruses (HPV) are a major etiologic driver of PSCC. However, the molecular details of the underlying carcinogenesis are understudied because of rare clinical specimens and missing cell lines. Here, we investigated if the expression of high-risk HPV16 oncogenes causes an augmentation of the Wnt pathway using unique HPV-positive penile cancer (PeCa) cell lines in monolayer and organotypic 3D raft cultures as well as tissue micro arrays containing clinical tissue specimens. The HPV oncoproteins enhanced the expression of Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) and the HPV-positive PeCa cells expressed a signature of Wnt target and stemness-associated genes. However, the notable lack of nuclear β-catenin in vitro and in situ raised the question if the enhanced expression of Wnt pathway factors is tantamount to an active Wnt signaling. Subsequent TOP-flash reporter assays revealed Wnt signaling as absent and not inducible by respective Wnt ligands in PeCa cell lines. The HPV-positive PeCa cells and especially HPV-positive PeCa specimens of the tumor core expressed the Wnt antagonist and negative feedback-regulator Dickkopf1 (DKK1). Subsequent neutralization experiments using PeCa cell line-conditioned media demonstrated that DKK1 is capable to impair ligand-induced Wnt signaling. While gene expression analyses suggested an augmented and active canonical Wnt pathway, the respective signaling was inhibited due to the endogenous expression of the antagonist DKK1. Subsequent TMA stainings indicated Dkk1 as linked with HPV-positivity and metastatic disease progression in PeCa suggesting potential as a prognostic marker.     

Low NLRP3 expression predicts a better prognosis of colorectal cancer

Background: NOD-like receptor pyrin domain-3 (NLRP3) inflammasome activation is a double-edged sword in tumorigenesis. Whether NLRP3 is involved in the progression and prognosis of colorectal cancer (CRC) remains elucidated and is the focus of the present study.Methods: Immunohistochemistry (IHC) was applied on tissue microarray (TMA) to determine the expression of NLRP3 in CRC patients. All 100 patients were divided into the low NLRP3 group and the high NLRP3 group according to their NLRP3 IHC scoring. Additionally, CRC xenografts were established by injecting HCT116 or RKO cells subcutaneously into nude mice. Cell proliferation and apoptosis were determined in HCT116 cells after treatment with NLRP3 inhibitor MCC950.Results: NLRP3 expression was up-regulated in colon adenocarcinoma tissues compared with that in paracancerous tissues in CRC patients, HCT116 xenograft, and RKO xenograft. High NLRP3 level correlated with the advanced TNM classification of malignant tumors, the occurrence of distant metastasis, vascular invasion, and positive lymph nodes. Furthermore, Kaplan–Meier survival analysis revealed that a high NLRP3 level was associated with a low 5-year survival rate and even a low 10-year survival rate. Moreover, the multivariable Cox proportional hazards regression model implied that NLRP3 expression level was an independent risk factor for CRC prognosis. Inhibition of NLRP3 by MCC950 suppressed cell proliferation, induced cell apoptosis, and decreased mRNA levels of interleukin 1β (IL1β) and interleukin 18 (IL18) in HCT116 cells.Conclusions: High level of NLRP3 predicts poor survival in CRC patients. NLRP3 is a putative prognostic biomarker and a potential therapeutic target in CRC treatments.     

MicroRNA‐181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2

γδ T cells are a conserved population of lymphocytes that contributes to anti‐tumor responses through its overt type 1 inflammatory and cytotoxic properties. We have previously shown that human γδ T cells acquire this profile upon stimulation with IL‐2 or IL‐15, in a differentiation process dependent on MAPK/ERK signaling. Here, we identify microRNA‐181a as a key modulator of human γδ T cell differentiation. We observe that miR‐181a is highly expressed in patients with prostate cancer and that this pattern associates with lower expression of NKG2D, a critical mediator of cancer surveillance. Interestingly, miR‐181a expression negatively correlates with an activated type 1 effector profile obtained from in vitro differentiated γδ T cells and miR‐181a overexpression restricts their levels of NKG2D and TNF‐α. Upon in silico analysis, we identify two miR‐181a candidate targets, Map3k2 and Notch2, which we validate via overexpression coupled with luciferase assays. These results reveal a novel role for miR‐181a as critical regulator of human γδ T cell differentiation and highlight its potential for manipulation of γδ T cells in next‐generation immunotherapies.     

Glutaminolysis and autophagy in cancer

The remarkable metabolic differences between cancer cells and normal cells result in the potential for targeted cancer therapy. The upregulation of glutaminolysis provides energetic advantages to cancer cells. The recently described link between glutaminolysis and autophagy, mediated by MTORC1, may constitute an attractive target for therapeutic strategies. A combination of therapies targeting simultane-ously cell signaling, cancer metabolism, and autophagy can solve therapy resistance and tumor relapse problems, commonly observed in patients treated with most of the current targeted therapies. In this review we summarize the mechanistic link between glutaminolysis and autophagy, and discuss the impacts of these processes on cancer progression and the potential for therapeutic intervention.     

PPARγ activation induces autophagy in breast cancer cells

It has been previously shown that PPARγ ligands induce apoptotic cell death in a variety of cancer cells. Given the evidence that these ligands have a receptor-independent function, we further examined the specific role of PPARγ activation in this biological process. Surprisingly, we failed to demonstrate that MDA-MB-231 breast cancer cells undergo apoptosis when treated with sub-saturation doses of troglitazone and rosiglitazone, which are synthetic PPARγ ligands. Acridine orange (AO) staining showed acidic vesicular formation within ligand-treated cells, indicative of autophagic activity. This was confirmed by autophagosome formation as indicated by redistribution of LC3, an autophagy-specific protein, and the appearance of double-membrane autophagic vacuoles by electron microscopy following exposure to ligand. To determine the mechanism by which PPARγ induces autophagy, we transduced primary mammary epithelial cells with a constitutively active mutant of PPARγ and screened gene expression associated with PPARγ activation by genome-wide array analysis. HIF1α and BNIP3 were among 42 genes up-regulated by active PPARγ. Activation of PPARγ induced HIF1α and BNIP3 protein and mRNA abundance. HIF1α knockdown by shRNA abolished the autophagosome formation induced by PPARγ activation. In summary, our data shows a specific induction of autophagy by PPARγ activation in breast cancer cells providing an understanding of distinct roles of PPARγ in tumorigenesis.     

LRP5 promotes cancer stem cell traits and chemoresistance in colorectal cancer

The overactivation of canonical Wnt/β‐catenin pathway and the maintenance of cancer stem cells (CSCs) are essential for the onset and malignant progression of most human cancers. However, their regulatory mechanism in colorectal cancer (CRC) has not yet been well demonstrated. Low‐density lipoprotein receptor‐related protein 5 (LRP5) has been identified as an indispensable co‐receptor with frizzled family members for the canonical Wnt/β‐catenin signal transduction. Herein, we show that activation of LRP5 gene promotes CSCs‐like phenotypes, including tumorigenicity and drug resistance in CRC cells, through activating the canonical Wnt/β‐catenin and IL‐6/STAT3 signalling pathways. Clinically, the expression of LRP5 is upregulated in human CRC tissues and closely associated with clinical stages of patients with CRC. Further analysis showed silencing of endogenous LRP5 gene is sufficient to suppress the CSCs‐like phenotypes of CRC through inhibiting these two pathways. In conclusion, our findings not only reveal a regulatory cross‐talk between canonical Wnt/β‐catenin signalling pathway, IL‐6/STAT3 signalling pathway and CD133‐related stemness that promote the malignant behaviour of CRC, but also provide a valuable target for the diagnosis and treatment of CRC.     

Curcumin inhibits hypoxia-induced migration in K1 papillary thyroid cancer cells

Curcumin, traditionally used as food and medicinal purposes, has recently been reported to have protective efficacy against hypoxia. Hypoxia is one of the important reactive factors in tumor metastasis, which is a key problem in clinical thyroid cancer therapy. In present study, we investigate the anti-metastatic effect of curcumin on the K1 papillary thyroid cancer cells as well as its potential mechanisms. The results show that curcumin effectively inhibits hypoxia-induced reactive oxygen species (ROS) upregulation and significantly decreases the mRNA and protein expression levels of hypoxia-inducible factor-1α (HIF-1α) in K1 cells. Curcumin also decreases the DNA binding ability of HIF-1α to hypoxia response element (HRE). Furthermore, curcumin enhances E-cadherin expression, inhibits metalloproteinase-9 (MMP-9) enzyme activity, and weakens K1 cells migration under hypoxic conditions. In summary, these results indicate that curcumin possesses a potent anti-metastatic effect and might be an effective tumoristatic agent for the treatment of aggressive papillary thyroid cancers.