Research progress concerning m6A methylation and cancer

N6-methyladenosine (m⁶A) methylation is a type of methylation modification on RNA molecules, which was first discovered in 1974, and has become a hot topic in life science in recent years. m⁶A modification is an epigenetic regulation similar to DNA and histone modification and is dynamically reversible in mammalian cells. This chemical marker of RNA is produced by m⁶A ‘writers’ (methylase) and can be degraded by m⁶A ‘erasers’ (demethylase). Methylated reading protein is the ‘reader’, that can recognize the mRNA containing m⁶A and regulate the expression of downstream genes accordingly. m⁶A methylation is involved in all stages of the RNA life cycle, including RNA processing, nuclear export, translation and regulation of RNA degradation, indicating that m⁶A plays a crucial role in RNA metabolism. Recent studies have shown that m⁶A modification is a complicated regulatory network in different cell lines, tissues and spatio-temporal models, and m⁶A methylation is associated with the occurrence and development of tumors. The present review describes the regulatory mechanism and physiological functions of m⁶A methylation, and its research progress in several types of human tumor, to provide novel approaches for early diagnosis and targeted treatment of cancer.     

Decreased RNA m6A methylation enhances the process of the epithelial mesenchymal transition and vasculogenic mimicry in glioblastoma

RNA N⁶-methyladenosine (m⁶A) modification is gradually thought to be an active participant in the considerable biological processes of glioblastoma (GB), providing us with a novel insight for exploring this disease. However, the role of RNA m⁶A modification during the epithelial mesenchymal transition (EMT) or vasculogenic mimicry (VM) progression has not been investigated in GB. Here we performed a research to validate the impact exerted by AlkB homolog 5 (ALKBH5), one of “erasers” for RNA m⁶A and methyltransferase-like 3 (METTL3) which adds m⁶A modification to the RNAs on the progression of EMT and VM in GB. In this study, we demonstrate that the m⁶A levels of RNAs were reduced in GB cells and glioma tissues. Patients with high mRNA expression of ALKBH5 acquired relatively shorter median overall survival (OS) time, while patients with relatively high expression of MEETL3 prolonged their disease free survival. ALKBH5 enhanced GB cell proliferation and influenced cell cycle in vitro. Decreased RNA m⁶A methylation enhanced the progression of the EMT and VM in glioblastoma cells. ALKBH5 strengthened glioblastoma growth and enhanced the EMT and VM process of glioblastoma in vivo. Our study uncovers that RNA m⁶A methylation suppresses the process of EMT and VM in glioblastoma, providing a new perspective to seek for a potential therapeutic target for GB.     

N6-methyladenosine (m6A) RNA modification in tumor immunity

Growing evidence supports that cancer progression is closely associated with the tumor microenvironment and immune evasion. Importantly, recent studies have revealed the crucial roles of epigenetic regulators in shaping the tumor microenvironment and restoring immune recognition. N⁶-methyladenosine (m⁶A) modification, the most prevalent epigenetic modification of mammalian mRNAs, has essential functions in regulating the processing and metabolism of its targeted RNAs, and therefore affects various biological processes including tumorigenesis and progression. Recent studies have demonstrated the critical functions and molecular mechanisms underlying abnormal m⁶A modification in the regulation of tumor immunity. In this review, we summarize recent research progress in the potential roles of m⁶A modification in tumor immunoregulation, with a special focus on the anti-tumor processes of immune cells and involvement in immune-associated molecules and pathways. Furthermore, we review current knowledge regarding the close correlation between m⁶A-related risk signatures and the tumor immune microenvironment landscape, and we discuss the prognostic value and therapeutic efficacy of m⁶A regulators in a variety of cancer types.     

miR-146b-5p mediates p16-dependent repression of IL-6 and suppresses paracrine procarcinogenic effects of breast stromal fibroblasts

Increasing evidence support the critical roles of active stromal fibroblasts in breast cancer development and spread. However, the mediators and the mechanisms of regulation are still not well defined. We have shown here that the tumor suppressor p16^INK4A protein inhibits the pro-carcinogenic effects of breast stromal fibroblasts through repressing the expression/secretion of IL-6. Indeed, p16^INK4A suppresses IL-6 at the mRNA and protein levels. This effect is mediated trough miR-146b-5p, which inhibits IL-6 expression through a specific sequence at the IL-6 3′UTR. In addition, we present clear evidence that miR-146b-5p inhibition is sufficient to transactivate breast stromal fibroblasts, which promote epithelial-to-mesenchymal-transition in breast cancer cells in a paracrine manner. By contrast, ectopic expression of miR-146b-5p in active fibroblasts abrogated their pro-carcinogenic effects. The physiological importance of miR-146b-5p inhibition was revealed by showing that the levels of pre-miR-146b-5p as well as its mature form are reduced in cancer-associated fibroblasts as compared with their normal adjacent counterparts from cancer-free tissues isolated from the same patients. Interestingly, treatment of active breast stromal fibroblasts with curcumin increased the level of the p16^INK4A coding CDKN2A mRNA and miR-146b-5p and suppressed IL-6, which confirms the repressive effect of these two tumor suppressor molecules on IL-6, and shows the possible “normalization” of cancer-related active fibroblasts. These results show that miR-146b-5p has non-cell-autonomous tumor suppressor function through inhibition of IL-6, suggesting that targeting this microRNA in breast stromal fibroblasts could be of great therapeutic value.     

ImmunoPET of the differential expression of CD146 in breast cancer

With advancement in antibody engineering, the development and characterization of new cancer-specific molecular targets are in the forefront of this PET-antibody combination “revolution”. Overexpression of CD146 in different types of tumors, including breast tumor, has been associated with tumor progression and poor prognosis. Non-invasive detection of CD146 with a monoclonal antibody may provide a noninvasive diagnostic tool with high specificity and accountability. Methods: Herein, we have developed a CD146-specific monoclonal antibody (YY146), radiolabeled it with ⁵²Mn and ⁸⁹Zr and identified its capability in acting as a non-invasive imaging agent that specific targets CD146 in different murine breast cancer models. CD146 expression was first screened in different breast tumor cell lines through Western Blot and confirmed its binding ability to YY146 using Flow Cytometry. Serial immunoPET images were carried out after intravenous administration of ⁵²Mn or ⁸⁹Zr labeled YY146. In addition, we also performed in vivo fluorescence imaging in animals injected with YY146 conjugated with Cy5.5. Results: Western Blot results show that MDA-MB-435 cell line had greater levels of CD146 expression when compared to the other cell lines investigated. Flow cytometry confirmed binding ability of YY146. PET images revealed well correlated uptake between tumor uptake and CD146 expression levels, confirmed by biodistribution studies and fluorescence imaging. Conclusion: PET imaging, for up to 7 days, of mice bearing three different breast tumors were carried out and revealed radiotracer uptake in tumors that strongly (r² = 0.98, P < 0.01), correlated with CD146 expression levels, as confirmed by in vitro and ex vivo studies.     

Lecithin: Cholesterol Acyltransferase and Na+-K+-ATPase Activity in Patients with Breast Cancer

Purpose

The aim of this study was to determine whether plasma lecithin:cholesterol acyltransferase (pLCAT) and erythrocyte membrane Na⁺-K⁺-ATPase ase (emNaKATPs) activity have a correlation in breast cancer. This study compared these parameters at time points before and after treatment with radiotherapy.

Methods

The levels of pLCAT and emNaKATPs were assessed in 30 patients with breast carcinoma and 20 control subjects. While emNaKATPs was measured with spectrophotometric method, pLCAT levels was measured using a specific enzyme-linked immunosorbent assay.

Results

pLCAT levels, both before and after radiotherapy, were found to be decreased in breast cancer patients than in the controls groups (p<0.001 and p<0.001, respectively). Also, pLCAT levels after radiotherapy were found to be decreased in breast cancer patients than the pLCAT levels before radiotherapy (p<0.001). The emNaKATPs activity were higher in the control group than in the breast cancer patients before/after radiotherapy (RT) (p<0.001 and p<0.001, respectively). At the same time, emNaKATPs activity before RT was higher in the breast cancer patients than emNaKATPs activity after RT (p<0.001). There was a significant correlation between pLCAT and emNaKATPs activity in breast cancer patients receiving radiotherapy (r=0.63, p<0.001), but no correlation between in breast cancer patients before RT and control group (r=0.023, p>0.05).

Conclusion

The results of the present study demonstrated that decreased pLCAT and emNaKATPs activity levels in breast cancer patients after/before RT than control group. In addition, decreased emNaKATPs activity in breast cancer patients receiving radiotherapy may be due to decreased pLCAT concentrations and RT beam. In our opinion, altered activities of pLCAT and emNaKATPs are linked to the treatment effect of radiotherapy. These data may clarify the development of cell membrane dysfunction and lipid metabolism in breast cancer patients receiving radiotherapy.     

The clinical significance of 99mTc-MIBI breast imaging in the diagnosis of early breast cancer

Objective: To find an effective, sensitive, specific and noninvasive diagnostic method of breast cancer. Methods: 109 masses of 102 patients with breast lesions smaller than 2 cm in diameter were divided into three groups to undergo ^99mTc-MIBI imaging and compared with the results of pathology examination. 20 cases without breast lesions were selected as control. Abnormal condensation of ^99mTc-MIBI in the breast reaching 10% higher than that in the counterpart of the healthy breast was regarded as positive. Results: Of 32 breast cancers, positive imaging appeared in 25. Negative imaging were found in 31 of 38 benign breast lesions. Of 39 occult breast lesions, positive imaging appeared in 6 and 3 of them were breast cancer, 2 of 3 patients with slightly increased ^99mTc-MIBI imaging threshold were breast cancer also. No positive imaging was found in the control group. The diagnostic accuracy, sensitivity, specificity, positive predictive value, negative predictive value of ^99mTc-MIBI was 88.4%, 89.2%, 88.0%, 75.0% and 95.3%, respectively. Conclusion: ^99mTc-MIBI imaging had higher sensitivity and accuracy in the diagnosis of breast cancer and differentiation between benign and malignant breast lesions. It could provide useful information for the diagnosis of clinically suspected breast cancer. Biography: Ren Chang-cai, (1951–), associate professor of general surgery, majors in breast and thyroid diseases, especially skillful on diagnosis and treatment of early stage breast cancer and treatment of advanced breast cancer.     

Downregulation of m6A writer complex member METTL14 in bladder urothelial carcinoma suppresses tumor aggressiveness

N6‐methyladenosine (m⁶A) and its regulatory proteins have been associated with tumorigenesis in several cancer types. However, knowledge on the mechanistic network related to m⁶A in bladder cancer (BlCa) is rather limited, requiring further investigation of its functional role. We aimed to uncover the biological role of m⁶A and related proteins in BlCa and understand how this influences tumor aggressiveness. N6‐adenosine‐methyltransferase catalytic subunit (METTL3), N6‐adenosine‐methyltransferase noncatalytic subunit (METTL14), protein virilizer homolog (VIRMA), and RNA demethylase ALKBH5 (ALKBH5) had significantly lower expression levels in BlCa compared to that in normal urothelium. METTL14 knockdown led to disruption of the remaining methyltransferase complex and a decrease in m⁶A abundance, as well as overall reduced tumor aggressiveness (decreased cell invasion and migration capacity and increased apoptosis). Furthermore, in vivo, METTL14 knockdown caused tumor size reduction. Collectively, we propose methyltransferase METTL14 as a key component for m⁶A RNA deposit and that it is closely related to BlCa progression, playing an important role in tumor aggressiveness. These data contribute to a better understanding of the m⁶A writer complex, which might constitute an appealing therapeutic target.     

Release of Ca2+ from the endoplasmic reticulum and its subsequent influx into mitochondria trigger celastrol-induced paraptosis in cancer cells

Celastrol, a triterpene extracted from the Chinese “Thunder of God Vine”, is known to have anticancer activity, but its underlying mechanism is not completely understood. In this study, we show that celastrol kills several breast and colon cancer cell lines by induction of paraptosis, a cell death mode characterized by extensive vacuolization that arises via dilation of the endoplasmic reticulum (ER) and mitochondria. Celastrol treatment markedly increased mitochondrial Ca²⁺ levels and induced ER stress via proteasome inhibition in these cells. Both MCU (mitochondrial Ca²⁺ uniporter) knockdown and pretreatment with ruthenium red, an inhibitor of MCU, inhibited celastrol-induced mitochondrial Ca²⁺ uptake, dilation of mitochondria/ER, accumulation of poly-ubiquitinated proteins, and cell death in MDA-MB 435S cells. Inhibition of the IP₃ receptor (IP₃R) with 2-aminoethoxydiphenyl borate (2-APB) also effectively blocked celastrol-induced mitochondrial Ca²⁺ accumulation and subsequent paraptotic events. Collectively, our results show that the IP₃R-mediated release of Ca²⁺ from the ER and its subsequent MCU-mediated influx into mitochondria critically contribute to celastrol-induced paraptosis in cancer cells.     

Effect of alternative temozolomide schedules on glioblastoma O6-methylguanine-DNA methyltransferase activity and survival

Background:

O⁶-methylguanine-DNA methyltransferase (MGMT) expression in glioblastoma correlates with temozolomide resistance. Dose-intense temozolomide schedules deplete MGMT activity in peripheral blood mononuclear cells; however, no published data exist evaluating the effect of temozolomide schedules on intracranial tumour MGMT activity.

Methods:

Human glioblastoma cells (GBM43) with an unmethylated MGMT promoter were implanted intracranially in immunodeficient rodents. Three weeks later, animals received temozolomide 200 mg m⁻² for 5 days (schedule A, standard dose) or 100 mg m⁻² for 21 days (schedule B, dose intense).

Results:

Tumour MGMT activity was depleted by day 6 in both treatment groups compared with baseline. O⁶-methylguanine-DNA methyltransferase activity returned to baseline by day 22 in the schedule A group, but remained suppressed in the schedule B group. By day 29, MGMT activity had returned to baseline in both groups. Mean tumour volume was significantly decreased compared with untreated controls with either schedule (P<0.01), although neither schedule was superior (P=0.60). Median survival was 64, 42, and 28 days for schedule A, schedule B, and no drug, respectively (P<0.001 A or B vs control, P=NS A vs B).

Conclusions:

Dose-intense temozolomide prolongs tumour MGMT activity depletion compared with standard dosing, however, survival was not improved in this model.     

18F-DCFPyL (PSMA) PET in the Management of Men with Biochemical Failure after Primary Therapy: Initial Clinical Experience of an Academic Cancer Center

Purpose: To describe the initial experience of an academic center using ¹⁸F-DCFPyL PET in managing men with recurrent prostate cancer. Materials & Methods: This prospective, single-arm IRB-approved study included men with biochemical failure after primary therapy for prostate cancer and negative/equivocal CT and bone scintigraphy who were candidates for salvage therapy, as determined by a multidisciplinary panel of experts. ¹⁸F-DCFPyL PET was assessed for the presence and extent of recurrence: local, oligometastatic (≤4), or extensive. Post-PET management and clinical outcome, including PSA response, was documented. For patients who received PET-directed ablative therapies, response was categorized as “complete” if PSA became undetectable or “favorable” if PSA decreased ≥50%. Results: Forty-seven men with biochemical failure after radical prostatectomy (n = 29), primary radiotherapy (n = 15) or focal tumor ablation (n = 3) were included. PET was positive in (43/47) 91.5%, including local recurrence in (9/47) 19.2%; oligometastatic disease in (16/47) 34%; and extensive metastatic disease in (18/47) 38.3%. PET-directed focal ablative therapies without systemic therapy were given to (13/29) 44.8% of patients without extensive metastases on PET with a mean PSA response of 69% (median, 74.5%; range: 35–100). Favorable biochemical response was observed in (10/13) 76.9% of patients with limited recurrence on PET, and in 23.1% (3/13), there was complete response. Conclusion: ¹⁸F-DCFPyL PET was positive in >90% of patients with biochemical failure. For those with limited recurrence, PSMA PET-directed local ablative therapies resulted in favorable outcome in more than 3 in 4 patients, and in nearly a quarter of them, there was complete biochemical response.     

Monocytes and macrophages,implications for breast cancer migration and stem cell-like activity and treatment

Macrophages are a major cellular constituent of the tumour stroma and contribute to breast cancer prognosis. The precise role and treatment strategies to target macrophages remain elusive. As macrophage infiltration is associated with poor prognosis and high grade tumours we used the THP-1 cell line to model monocyte-macrophage differentiation in co-culture with four breast cancer cell lines (MCF7, T47D, MDA-MB-231, MDA-MB-468) to model in vivo cellular interactions. Polarisation into M1 and M2 subtypes was confirmed by specific cell marker expression of ROS and HLA-DR, respectively. Co-culture with all types of macrophage increased migration of ER-positive breast cancer cell lines, while M2-macrophages increased mammosphere formation, compared to M1-macrophages, in all breast cancer cells lines. Treatment of cells with Zoledronate in co-culture reduced the “pro-tumourigenic” effects (increased mammospheres/migration) exerted by macrophages. Direct treatment of breast cancer cells in homotypic culture was unable to reduce migration or mammosphere formation.Macrophages promote “pro-tumourigenic” cellular characteristics of breast cancer cell migration and stem cell activity. Zoledronate targets macrophages within the microenvironment which in turn, reduces the “pro-tumourigenic” characteristics of breast cancer cells. Zoledronate offers an exciting new treatment strategy for both primary and metastatic breast cancer.     

Phase I trial of temozolomide plus O6-benzylguanine 5-day regimen with recurrent malignant glioma

This phase I clinical trial conducted with patients who had recurrent or progressive malignant glioma (MG) was designed to determine the maximum tolerated dose (MTD) and toxicity of three different 5-day dosing regimens of temozolomide (TMZ) in combination with O⁶-benzylguanine (O⁶-BG). Both TMZ and O⁶-BG were administered on days 1–5 of a 28-day treatment cycle. A bolus infusion of O⁶-BG was administered at 120 mg/m² over 1 h on days 1, 3, and 5, along with a continuous infusion of O⁶-BG at 30 mg/m²/day. TMZ was administered at the end of the first bolus infusion of O⁶-BG and then every 24 h for 5 days during the continuous infusion of O⁶-BG. Patients were accrued to one of three 5-day dosing regimens of TMZ. Twenty-nine patients were enrolled into this study. The dose-limiting toxicities (DLTs) were grade 4 neutropenia, leukopenia, and thrombocytopenia. The MTD for TMZ for the three different 5-day dosing schedules was determined as follows: schedule 1, 200 mg/m² on day 1 and 50 mg/m²/day on days 2–5; schedule 2, 50 mg/m²/day on days 1–5; and schedule 3, 50 mg/m²/day on days 1–5 while receiving pegfilgrastim. Thus, the 5-day TMZ dosing schedule that maximized the total dose of TMZ when combined with O⁶-BG was schedule 1. This study provides the foundation for a phase II trial of O⁶-BG in combination with a 5-day dosing schedule of TMZ in TMZ-resistant MG.     

YM155 potently triggers cell death in breast cancer cells through an autophagy-NF-kB network

Specific overexpression in cancer cells and evidence of oncogenic functions make Survivin an attractive target in cancer tharapy. The small molecule compound YM155 has been described as the first “Survivin suppressant” but molecular mechanisms involved in its biological activity and its clinical potential remain obscure. We herein show that YM155 exerts single agent toxicity on primary breast cancer cells grown in an ex vivo assay preserving tumor microenvironment. In vitro assays indicate that YM155 more efficiently triggers cell death in breast cancer cells (including these with stem-cell like properties) than in non tumorigenic mammary cells. YM155-induced cell death is critically dependent on autophagy and NF-kB but independent of p53 and it coïncides with DNA damage an a DNA damage response in p53-proficient cells. Our results point out a crosstalk between NF-KB and autophagy controlling YM155-induced death in breast cancer cells and argue for the potential use of YM155 as a genotoxic agent in breast cancer therapy.     

Tc-sestamibi radio-guided surgery of loco-regional Iodine-negative recurrent thyroid cancer

Aim

We report here our experience in a larger series of differentiated thyroid cancer (DTC) patients who had been treated by ^99mTc-sestamibi radio-guided surgery (RGS) for ¹³¹Iodine (¹³¹I)-negative loco-regional recurrent disease.

Methods

Fifty-eight patients with loco-regional ¹³¹I-negative recurrent disease from DTC were studied with ^99mTc-sestamibi directed RGS using a hand-held 11-mm gamma probe as an intra-operative detector. Patients were selected for RGS on the basis of (a) progressive increase of serum thyroglobulin (Tg) levels after first treatment during follow-up, (b) negative high dose (100 mCi, 3.7 GBq) ¹³¹I whole-body scan, and (c) positive pre-operative ^99mTc-sestamibi scintigraphy for the presence of loco-regional recurrent disease. There were 41 papillary (1 “tall” cell variant), 13 follicular and 4 Hürthle cells tumours. In 14 patients thyroid cancer recurred in the thyroid bed while cervical lymph node metastases were found in 37 patients, and 7 patients had recurrent disease both in the thyroid bed and in cervical lymph nodes.

Results

At bilateral neck exploration, 147 metastastic foci ranging from 4 mm to 51 mm in largest diameter (mean tumour diameter = 17.3 ± 9.5 mm) were removed. Eighty-five of them (58%) had been pre-operatively identified at ^99mTc-sestamibi scintigraphy. After RGS, serum Tg levels normalised in 43 of 58 patients (serum Tg < 2 ng/ml – they were considered disease-free), serum Tg remained slightly increased in 12 patients without evidence of metastatic disease at scintigraphic and radiologic imaging (serum Tg < 10 ng/mg – they were considered living with microscopic disease), while serum Tg significantly increased up to values > 900 ng/ml in 3 patients who developed lung metastases. The mean lesion to background ^99mTc-sestamibi uptake ratios decreased in all 58 patients (p < 0.0001). Post-surgical follow-up ranged 6–72 months (mean ± SD = 29.6 ± 13.5 months). The operating surgeon assessed RGS as very useful in 14 patients in whom metastatic foci were embedded in fibrotic tissues or located behind blood vessels, useful in 22 patients, moderately useful 17 patients and not useful in 5 patients.

Conclusion

Our data suggest that a ^99mTc-sestamibi intra-operative gamma probe can be used to identify and guide resection of recurrent loco-regional tumour in DTC patients with ¹³¹I-negative loco-regional metastatic foci.     

m6A RNA甲基化在非小细胞肺癌中的研究进展

m⁶A修饰是真核生物mRNA中最丰富的修饰之一，该过程受m⁶A甲基转移酶和去甲基化酶的共同调控。m⁶A修饰后的RNA能够被m⁶A识别蛋白特异性识别并结合，进而介导RNA的剪接、成熟、出核、降解和翻译等。目前国内外对于m⁶A修饰及其相关蛋白如何参与非小细胞肺癌发生发展的研究，主要集中于细胞恶性增殖、迁移、侵袭、转移和耐药等方面。m⁶A修饰相关蛋白在肺癌组织标本和血液循环肿瘤细胞（circulating tumor cell, CTC）中表达异常，有望成为肺癌诊断和预后判断的潜在分子标志物。本文围绕m⁶A修饰相关蛋白的组成、作用方式、在非小细胞肺癌恶性进展中的生物学功能，以及针对m⁶A修饰的靶向治疗等方面的研究进展进行综述，旨在为非小细胞肺癌的早期临床诊断和靶向药物的开发提供新思路。