Akt isoforms differentially provide for chemoresistance in prostate cancer

Objective:Early prostate cancer micrometastatic foci undergo a mesenchymal to epithelial reverting transition,not only aiding seeding and colonization,but also rendering the tumor cells generally chemoresistant.We previously found that upregulated E-cadherin in the epithelial micrometastases activated canonical survival pathways,including PI3K-Akt,that protected the tumor cells from death;however,the extent of protection from blocking the pathway in its entirety was modest,because different isoforms may have alternately affected cell functioning.Here,we characterized Akt isoform expressions in primary and metastatic prostate cancers,as well as their individual contributions to chemoresistance.Methods:Akt isoforms and E-cadherin were manipulated with drugs,knocked down,and over expressed.Tumor cell killing was determined in vitro and in vivo.Overall survival was calculated from patient records and specimens.Results:Pan-Akt inhibition sensitized tumor cells to chemotherapy,and specific blockade of Akt1 or/and Akt2 caused cells to be more chemoresponsive.Overexpression of Akt3 induced apoptosis.A low dose of Akt1 or Akt2 inhibitor enabled standard chemotherapies to significantly eradicate metastatic prostate tumors in a mouse model,acting as chemosensitizers.In human specimens,we found Akt1 and Akt2 positively correlated,whereas Akt3 inversely correlated,with the overall survival of prostate cancer patients.Akt1high/Akt2high/Akt3low tumors had the worst outcomes.Conclusions:E-cadherin-induced activation of Akt1/2 isoforms was the essential mechanism of chemoresistance,whereas Akt3 made cells more fragile.These findings emphasized the need to target Akt1/2,rather than pan-Akt,as a rational therapeutic approach.

Transcriptome analysis creates a new era of precision medicine for managing recurrent hepatocellular carcinoma

The high incidence of hepatocellular carcinoma(HCC)recurrence negatively impacts outcomes of patients treated with curative intent despite advances in surgical techniques and other locoregional liver-targeting therapies.Over the past few decades,the emergence of transcriptome analysis tools,including real-time quantitative reverse transcription PCR,microarrays,and RNA sequencing,has not only largely contributed to our knowledge about the pathogenesis of recurrent HCC but also led to the development of outcome prediction models based on differentially expressed gene signatures.In recent years,the single-cell RNA sequencing technique has revolutionized our ability to study the complicated crosstalk between cancer cells and the immune environment,which may benefit further investigations on the role of different immune cells in HCC recurrence and the identification of potential therapeutic targets.In the present article,we summarized the major findings yielded with these transcriptome methods within the framework of a causal model consisting of three domains:primary cancer cells;carcinogenic stimuli;and tumor microenvironment.We provided a comprehensive review of the insights that transcriptome analyses have provided into diagnostics,surveillance,and treatment of HCC recurrence.

Inhibition of Cdk5 increases osteoblast differentiation and bone mass and improves fracture healing

Identification of regulators of osteoblastogenesis that can be pharmacologically targeted is a major goal in combating osteoporosis,a common disease of the elderly population. Here, unbiased kinome RNAi screening in primary murine osteoblasts identified cyclin-dependent kinase 5(Cdk5) as a suppressor of osteoblast differentiation in both murine and human preosteoblastic cells. Cdk5 knockdown by si RNA, genetic deletion using the Cre-lox P system, or inhibition with the small molecule roscovitine enhanced osteoblastogenesis in vitro. Roscovitine treatment significantly enhanced bone mass by increasing osteoblastogenesis and improved fracture healing in mice. Mechanistically, downregulation of Cdk5 expression increased Erk phosphorylation, resulting in enhanced osteoblast-specific gene expression. Notably, simultaneous Cdk5 and Erk depletion abrogated the osteoblastogenesis conferred by Cdk5 depletion alone, suggesting that Cdk5 regulates osteoblast differentiation through MAPK pathway modulation. We conclude that Cdk5 is a potential therapeutic target to treat osteoporosis and improve fracture healing.

Evaluation of Daily Tumor Motion by Measuring Fiducial Length on CBCT Images in Pancreatic Stereotactic Body Radiation Therapy

We investigated the feasibility of measuring daily fiducial length on cone-beam computed tomography (CBCT) images to assess the variation in daily tumor motion for pancreatic SBRT. Motion data for fifty pancreatic SBRT patients with fiducials were analyzed retrospectively to determine the tumor motion statistics. We also performed a phantom study which involved motion analysis of three gold fiducials placed around a solid target inside the Quasar Phantom as a function of variable tumor motion and breathing period. The end-exhalation CT-50 images were compared with the CBCT images acquired prior to treatment delivery on a TrueBeam STx linear accelerator. Sinusoidal tumor motion and patients’ breathing files acquired from a Varian-RPM system were used to simulate patients’ breathing patterns. The fiducial length was measured to determine its correlation with tumor motion. Patient tumor motions along the superior-inferior (SI), anterior-posterior (AP), and left-right (LR) directions were found to be 0.7 ± 0.4 cm, 0.2 ± 0.3 cm, and 0.1 ± 0.2 cm, respectively. Average breathing period was 4.3 ± 0.8 seconds. For sinusoidal and patients’ breathing patterns, a significant correlation was observed between the fiducial length and tumor motions with R2 of 0.99. However, fiducial length was found to be independent of the variation in breathing periods. This work suggests that measuring the fiducial length on daily CBCT images could provide quantitative daily tumor motion for fiducial-based pancreatic SBRT. A timely decision to modify the motion management strategy could be made prior to daily treatment delivery.

Herbal formula enhances 5-fluorouracil activity through suppression of thymidylate synthase

Traditional Chinese herbal medicine(TCM)has been shown to enhance the efficacy of standard anticancer agents.However,there are only a limited number of well-controlled preclinical and clinical studies documenting the potential benefit of TCM.OBJECTIVE To identify biologically active formulas that were effective against colorectal cancer(CRC)by screening TCM formulas in in vitro and in vivo animal models.METHODS Cell growth assays,cell cycle analysis,immunoblot analysis and qRT-PCR were performed to investigate the mechanism(s)of action of the formulason human CRC cells.In vivo animal models were used to evaluate the antitumor activity of formulasalone and in combination with 5-FU.RESULTS We identified Huangqin Gegen Tang(HQGGT)which suppressed the in vivo growth of human CRC HT-29 xenografts.HQGGT significantly inhibited the growth of CRC cell lines.HQGGT enhanced the cytotoxicity of 5-FU against human 5-FU-resistant cells(H630R1)and mouse colon cancer cells(MC38).This synergy was the result of suppression of thymidylate synthase expression by HQGGT.HQGGT significantly enhanced the antitumor effect of 5-FU in mice bearing MC38 xenografts.Ongoing studies have identified Huangqin as the herb responsible for TS inhibi⁃tion.CONCLUSION These findings provide support for the potential role of HQGGT as a novel modulator of fluoropyrim⁃idine chemotherapy for CRC treatment.

PICOT promotes T lymphocyte proliferation by down-regulating cyclin D2 expression

The mammalian protein kinase C-interacting cousin of thioredoxin(PICOT;also termed glutaredoxin 3)is a multi-domain monothiol glutaredoxin that is involved in a wide variety of signaling pathways and biological processes.PICOT is required for normal and transformed cell growth and is critical for embryonic development.Recent studies in T lymphocytes demonstrated that PICOT can translocate to the nucleus and interact with embryonic ectoderm development,a polycomb group protein and a core component of the polycomb repressive complex 2,which contributes to the maintenance of transcriptional repression and chromatin remodeling.Furthermore,PICOT was found to interact with chromatin-bound embryonic ectoderm development and alter the extent of histone 3 lysine 27 trimethylation at the promoter region of selected polycomb repressive complex 2 target genes.PICOT knockdown in Jurkat T cells led to increased histone 3 lysine 27 trimethylation at the promoter region of CCND2,a cell cycle-regulating gene which encodes the cyclin D2 protein.As a result,the expression levels of CCND2 mRNA and protein levels were reduced,concomitantly with inhibition of the cell growth rate.Analysis of multiple data sets from the Cancer Genome Atlas revealed that a high expression of PICOT correlated with a low expression of CCND2 in a large number of human cancers.In addition,this parameter correlated with poor patient survival,suggesting that the ratio between PICOT/CCND2 mRNA levels might serve as a predictor of patient survival in selected types of human cancer.

Leelamine suppresses cMyc expression in prostate cancer cells in vitro and inhibits prostate carcinogenesis in vivo

Aim:Leelamine(LLM)inhibits the growth of human prostate cancer cells but the underlying mechanism is not fully understood.The present study was undertaken to determine the effect of LLM on cMyc,which is overexpressed in a subset of human prostate cancers.Methods:The effect of LLM on cMyc expression and activity was determined by western blotting/confocal microscopy and luciferase reporter assay,respectively.A transgenic mouse model of prostate cancer(Hi-Myc)was used to determine the chemopreventive efficacy of LLM.Results:Exposure of androgen-sensitive(LNCaP)and castration-resistant(22Rv1)human prostate cancer cells to LLM resulted in downregulation of protein and mRNA levels of cMyc.Overexpression of cMyc partially attenuated LLM-mediated inhibition of colony formation,cell viability,and cell migration in 22Rv1 and/or PC-3 cells.LLM treatment decreased protein levels of cMyc targets(e.g.,lactate dehydrogenase),however,overexpression of cMyc did not attenuate these effects.A trend for a decrease in the expression level of cMyc protein was discernible in 22Rv1 xenografts from LLM-treated mice compared with control mice.LLM treatment(10 mg/kg body weight,5 times/week)was well-tolerated by Hi-Myc transgenic mice.The incidence of high-grade prostatic intraepithelial neoplasia,adenocarcinoma in situ,and microinvasion were lower in LLM-treated Hi-Myc mice but the difference was not statistically significant.Conclusion:The present study reveals that LLM inhibits cMyc expression in human prostate cancer cells in vitro but concentrations higher than 10 mg/kg may be required to achieve chemoprevention of prostate cancer.

Statins and endocrine resistance in breast cancer

Most breast cancers are hormone-receptor positive(HR+).However,more women eventually die from HR+breast cancer than from either HER2+or triple negative breast cancer.Endocrine therapies continue to be the mainstay of treatment.In 40%of these cases,recurrences in early-stage disease and progression in the metastatic setting are largely a function of the development of endocrine resistance.A multitude of mediators and pathways have been associated with endocrine resistance in breast cancer including the mevalonate pathway,which is integral to cholesterol biosynthesis.The mevalonate pathway and the downstream activation of associated cytoplasmic pathways including PI3K-AKT-mTOR and RAS-MEK-ERK have been known to affect cancer cell proliferation,cell survival,cell invasion,and metastasis.These are important mechanisms leading to the inevitable development of endocrine resistance in HR+breast cancer.Statins are a class of drugs that inhibits HMG-CoA reductase,an enzyme in the mevalonate pathway that plays a central role in cholesterol production.In vitro and in vitro studies suggest that the role of statins in blocking the mevalonate pathway effectively disrupts downstream pathways involved in estrogen receptor expression and cellular processes such as cell survival,proliferation,stress,cell cycle,inhibition of apoptosis,and autophagy.Overcoming these key mechanisms heralds a role for statins in the prevention of endocrine resistance.

Role of Receptor Interacting Protein(RIP)kinases in cancer

The Receptor Interacting Protein(RIP)kinase family consists of seven Serine/Threonine kinases,which plays a key signaling role in cell survival and cell death.Each RIP family member contains a conserved kinase domain and other domains that determine the specific kinase function through proteineprotein interactions.RIP1 and RIP3 are best known for their critical roles in necroptosis,programmed necrosis and a non-apoptotic inflammatory cell death process.Dysregulation of RIP kinases contributes to a variety of pathogenic conditions such as inflammatory diseases,neurological diseases,and cancer.In cancer cells,alterations of RIP kinases at genetic,epigenetic and expression levels are frequently found,and suggested to promote tumor progression and metastasis,escape of antitumor immune response,and therapeutic resistance.However,RIP kinases can be either pro-tumor or anti-tumor depending on specific tumor types and cellular contexts.Therapeutic agents for targeting RIP kinases have been tested in clinical trials mainly for inflammatory diseases.Deregulated expression of these kinases in different types of cancer suggests that they represent attractive therapeutic targets.The focus of this review is to outline the role of RIP kinases in cancer,highlighting potential opportunities to manipulate these proteins in cancer treatment.

A phaseⅡprospective trial of photobiomodulation therapy in limiting oral mucositis in the treatment of locally advanced head and neck cancer patients

Objective:This study aimed to compare the historical incidence rate of severe oral mucositis(OM)in head and neck cancer patients undergoing definitive concurrent chemoradiation therapy(CRT)versus a prospective cohort of patients with locally advanced head and neck squamous cell carcinoma(HNSCC)treated with prophylactic photobiomodulation therapy(PBMT).Methods:This US-based,institutional,single-arm,phaseⅡprospective clinical trial was initiated in 50 patients(age≥18 years,Karnofsky Performance Scale Index>60,with locally advanced HNSCC(excluding oral cavity)receiving definitive or adjuvant radiation therapy(RT)with concurrent platinum-based chemotherapy(CT).PBMT was delivered three times per week throughout RT utilizing both an intraoral as well extraoral delivery system.Primary outcome measure was incidence of severe OM utilizing both the National Cancer Institute Common Toxicity Criteria,version 4.0(NCI-CTCAE)Grade≥3 and the World Health Organization Mucositis Grading Scale(WHO)Grade≥3 versus historical controls;secondary outcome measures included time to onset of severe OM following therapy initiation.Results:At baseline,all patients included in final analysis(N=47)had OM Grade 0.Average RT and CT dose was(66.3±5.1)Gy and(486.1±106.8)mg/m ^(2),respectively.Severe OM was observed in 11 of 47 patients(23%,confidence interval:12,38).OM toxicity grade trended upward during treatment,reaching a maximum at 7 weeks(WHO:1.8 vs.NCI-CTCAE:1.7).Subsequently,OM grade returned to baseline 3 months following completion of RT.The mean time to onset of severe OM was(35±12)days.The mean time to resolution of severe OM was(37±37)days.Conclusions:Compared to historical outcomes,PBMT aides in decreasing severe OM in patients with locally advanced HNSCC.PBMT represents a minimally invasive,prophylactic intervention to decrease OM as a major treatment-related side effect.

Fabrication of 3D-printed molds for polydimethylsiloxane-based microfluidic devices using a liquid crystal display-based vat photopolymerization process:printing quality,drug response and 3D invasion cell culture assays

Microfluidic platforms enable more precise control of biological stimuli and environment dimensionality than conventional macroscale cell-based assays;however,long fabrication times and high-cost specialized equipment limit the widespread adoption of microfluidic technologies.Recent improvements in vat photopolymerization three-dimensional(3D)printing technologies such as liquid crystal display(LCD)printing offer rapid prototyping and a cost-effective solution to microfluidic fabrication.Limited information is available about how 3D printing parameters and resin cytocompatibility impact the performance of 3D-printed molds for the fabrication of polydimethylsiloxane(PDMS)-based microfluidic platforms for cellular studies.Using a low-cost,commercially available LCD-based 3D printer,we assessed the cytocompatibility of several resins,optimized fabrication parameters,and characterized the minimum feature size.We evaluated the response to both cytotoxic chemotherapy and targeted kinase therapies in microfluidic devices fabricated using our 3D-printed molds and demonstrated the establishment of flow-based concentration gradients.Furthermore,we monitored real-time cancer cell and fibroblast migration in a 3D matrix environment that was dependent on environmental signals.These results demonstrate how vat photopolymerization LCD-based fabrication can accelerate the prototyping of microfluidic platforms with increased accessibility and resolution for PDMS-based cell culture assays.

In vitro and in vivo evaluation of cerium oxide nanoparticles in respiratory syncytial virus infection

Respiratory syncytial virus(RSV)is the most common cause of viral bronchiolitis among children worldwide,yet there is no vaccine for RSV disease.This study investigates the potential of cube and sphere-shaped cerium oxide nanoparticles(CNP)to modulate reactive oxygen(ROS)and nitrogen(RNS)species and immune cell phenotypes in the presence of RSV infection in vitro and in vivo.Cube and sphere-shaped CNP were synthesized by hydrothermal and ultrasonication methods,respectively.Physico-chemical characterization confirmed the shape of sphere and cube CNP and effect of various parameters on their particle size distribution and zeta potential.In vitro results revealed that sphere and cube CNP differentially modulated ROS and RNS levels in J774 macrophages.Specifically,cube CNP significantly reduced RSV-induced ROS levels without affecting RNS levels while sphere CNP increased RSV-induced RNS levels with minimal effect on ROS levels.Cube CNP drove an M1 phenotype in RSV-infected macrophages in vitro by increasing macrophage surface expression of CD80 and CD86 with a concomitant increase in TNFαand IL-12p70,while simultaneously decreasing M2 CD206 expression.Intranasal administration of sphere and cube-CNP were well-tolerated with no observed toxicity in BALB/c mice.Notably,cube CNP preferentially accumulated in murine alveolar macrophages and induced their activation,avoiding enhanced uptake and activation of other inflammatory cells such as neutrophils,which are associated with RSV-mediated inflammation.In conclusion,we report that sphere and cube CNP modulate macrophage polarization and innate cellular responses during RSV infection.

Vinyl chloride accident unleashes a toxic legacy

A railroad accident on February 3,2023,led to the release and combustion of 115,580 gallons,equivalent to over 437,000 L,of vinyl chloride monomer(VCM)in East Palestine,Ohio[1].This monomer is used in polyvinyl chloride(PVC)production,and its burning produces additional toxins such as hydrochloric acid and lethal phosgene,known as a notorious chemical weapon during World War I[2].Acute exposure to these chemicals causes immediate adverse effects on local ecosystems,including the deaths of wild and farmed animals and pets.

Immunogenic effects of chemotherapyinduced tumor cell death

Emerging evidence suggests that the clinical success of conventional chemotherapy is not solely attributed to tumor cell toxicity,but also results from the restoration of immunosurveillance,which has been largely neglected in the past preclinical and clinical research.Antitumor immune response can be primed by immunogenic cell death(ICD),a type of cell death characterized by cell-surface translocation of calreticulin(CRT),extracellular release of ATP and high mobility group box 1(HMGB1),and stimulation of type I interferon(IFN)responses.Here we summarize recent studies showing conventional chemotherapeutics as ICD inducers,which are capable of modulating tumor infiltrating lymphocytes(TILs)and reactivating antitumor immunity within an immuno-suppressive microenvironment.Such immunological effects of conventional chemotherapy are likely critical for better prognosis of cancer patients.Furthermore,combination of ICD-inducing chemotherapeutics with immunotherapy is a promising approach for improving the clinical outcomes of cancer patients.

The oxidative DNA damage response： A review of research undertaken with Tsinghua and Xiangya students at the University of Pittsburgh

Endogenous stress and exogenous toxicants(chemicals and UV light) alter genetic information either directly or indirectly through the production of reactive oxygen species(ROS), thereby driving genomic instability in cells and promoting tumorigenesis. All living cells try to faithfully preserve and transmit their genomic information from one generation to the next using DNA repair mechanisms to repair oxidative DNA damage to prevent cancer or premature aging. Oxidative DNA damage comprises a mixture of DNA lesions including base damage, DNA single strand breaks(SSBs), and DNA double strand breaks(DSBs). This review summarizes some of the studies on DNA damage response at a defined genome locus that are performed by students from the Tsinghua University School of Medicine and the School of Medicine of Central South University(Xiangya Hospital) at the University of Pittsburgh School of Medicine. A summary of their work highlights the continuous contribution of the students to a particular research program and exemplifies the achievements of this China-U.S. collaborative training program.

中年含糖饮料摄入频率与老年认知障碍的关系

目的探究中年时期含糖饮料摄入频率与老年认知障碍风险的关系。方法对来自新加坡华族健康队列的16948名研究对象进行16~23年(平均20年)的随访,研究对象的含糖饮料摄入信息于基线调查获得(1993-1998年),研究对象的认知状态通过第3次随访期间(2014-2016年)在新加坡人群中校正过的简易智力状态检查量表获得。采用多元logistic线性回归模型分析含糖饮料摄入频率与认知障碍风险的关系。结果在纳入的研究对象中,2443名存在认知障碍。在控制了年龄、性别、籍贯、文化程度、生活方式(包括饮食因素)和疾病信息后,含糖饮料摄入频率与认知障碍风险关联无统计学意义。与几乎不喝碳酸饮料组相比,每周摄入碳酸饮料≥2次(OR=0.91,95%CI:0.77~1.08)、每周摄入1次(OR=1.00,95%CI:0.82~1.23)和每月摄入1~3次(OR=0.94,95%CI:0.80~1.09)的研究对象认知障碍发生风险差异无统计学意义(趋势性P=0.306)。与几乎不喝果(蔬)汁组相比,每周摄入果(蔬)汁≥2次(OR=1.03,95%CI:0.88~1.20)、每周摄入1次(OR=0.96,95%CI:0.82~1.12)和每月摄入1~3次(OR=0.94,95%CI:0.82~1.08)的研究对象的认知障碍发生风险均无统计学差异(趋势性P=0.930)。分层分析及敏感性分析对研究结果都没有实质改变。结论在平均含糖饮料摄入频率较低的新加坡籍华人中,中年时期含糖饮料摄入频率与老年认知障碍风险差异无统计学意义。

Transcriptional landscapes of de novo root regeneration from detached Arabidopsis leaves revealed by time-lapse and single-cell RNA sequencing analyses

Detached Arabidopsis thaliana leaves can regenerate adventitious roots,providing a platformfor studying de novo root regeneration(DNRR).However,the comprehensive transcriptional framework of DNRR remains elusive.Here,we provide a high-resolution landscape of transcriptome reprogramming from wound response to root organogenesis in DNRR and show key factors involved in DNRR.Time-lapse RNA sequencing(RNA-seq)of the entire leaf within 12 h of leaf detachment revealed rapid activation of jasmonate,ethylene,and reactive oxygen species(ROS)pathways in response towounding.Genetic analyses confirmed that ethylene andROSmay serve as wound signals to promoteDNRR.Next,time-lapse RNA-seq within 5 d of leaf detachment revealed the activation of genes involved in organogenesis,wound-induced regeneration,and resource allocation in the wounded region of detached leaves during adventitious rooting.Genetic studies showed that BLADE-ON-PETIOLE1/2,which control aboveground organs,PLETHORA3/5/7,which control root organogenesis,and ETHYLENE RESPONSE FACTOR115,which controlswound-induced regeneration,are involved in DNRR.Furthermore,single-cell RNA-seq data revealed gene expression patterns in thewounded region of detached leaves during adventitious rooting.Overall,our study not only provides transcriptome tools but also reveals key factors involved in DNRR from detached Arabidopsis leaves.