Novel Adiponectin Receptor Agonist Inhibits Cholangiocarcinoma via Adenosine Monophosphate-activated Protein Kinase.

BACKGROUND: Cholangiocarcinoma (CCA) has a poor prognosis and only limited palliative treatment options. The deficiency of adiponectin and adenosine monophosphate-activated protein kinase (AMPK) signaling was reported in several malignancies, but the alteration of these proteins in CCA is still unclear. OBJECTIVES: This study aimed to assess the role of adiponectin and AMPK signaling in CCA. Furthermore, AdipoRon, a novel adiponectin receptor (AdipoR) agonist, was evaluated in vitro and in vivo as a new anti-tumor therapy for CCA. METHODS: The expression of AdipoR1 and p-AMPKα in human tissue microarrays (TMAs) was evaluated by immunohistochemistry staining (IHC). The effect of 2-(4-Benzoylphenoxy)-N-[1-(phenylmethyl)- 4-piperidinyl]-acetamide (AdipoRon) was investigated in vitro with proliferation, crystal violet, migration, invasion, colony formation, senescence, cell cycle and apoptosis assays and in vivo using a CCA engineered mouse model (AlbCre/LSL-KRASG12D/p53L/L). RT-qPCR and western blot methods were applied to study molecular alterations in murine tissues. RESULTS: AdipoR1 and p-AMPKα were impaired in human CCA tissues, compared to adjacent non-tumor tissue. There was a positive correlation between the AdipoR1 and p-AMPKα levels in CCA tissues. Treatment with AdipoRon inhibited proliferation, migration, invasion and colony formation and induced apoptosis in a time- and dose-dependent manner in vitro(p<0.05). In addition, AdipoRon reduced the number of CCA and tumor volume, prolonged survival, and decreased metastasis and ascites in the treated group compared to the control group (p<0.05). CONCLUSIONS: AdipoR1 and p-AMPKα are impaired in CCA tissues, and AdipoRon effectively inhibits CCA in vitro and in vivo. Thus, AdipoRon may be considered as a potential anti-tumor therapy in CCA.

Progesterone Receptor Membrane Component 1 (PGRMC1) Modulates Tumour Progression, the Immune Microenvironment and the Response to Therapy in Glioblastoma.

Progesterone Receptor Membrane Component 1 (PGRMC1) is a tumour-promoting factor in several types of cancer but its role in brain tumours is poorly characterized thus far. Our study aimed to determine the effect of PGRMC1 on glioblastoma (GBM) pathophysiology using two independent cohorts of IDH wild-type GBM patients and stable knockdown GBM models. We found that high levels of PGRMC1 significantly predicted poor overall survival in both cohorts of GBM patients. PGRMC1 promoted the proliferation, anchorage-independent growth, and invasion of GBM cells. We identified Integrin beta-1 (ITGB1) and TCF 1/7 as potential members of the PGRMC1 pathway in vitro. The levels of ITGB1 and PGRMC1 also correlated in neoplastic tissues from GBM patients. High expression of PGRMC1 rendered GBM cells less susceptible to the standard GBM chemotherapeutic agent temozolomide but more susceptible to the ferroptosis inducer erastin. Finally, PGRMC1 enhanced Interleukin-8 production in GBM cells and promoted the recruitment of neutrophils. The expression of PGRMC1 significantly correlated with the numbers of tumour-infiltrating neutrophils also in tissues from GBM patients. In conclusion, PGRMC1 enhances tumour-related inflammation and promotes the progression of GBM. However, PGRMC1 might be a promising target for novel therapeutic strategies using ferroptosis inducers in this type of cancer.

PLOD2 Is a Prognostic Marker in Glioblastoma That Modulates the Immune Microenvironment and Tumor Progression.

This study aimed to investigate the role of Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase 2 (PLOD2) in glioblastoma (GBM) pathophysiology. To this end, PLOD2 protein expression was assessed by immunohistochemistry in two independent cohorts of patients with primary GBM (n1 = 204 and n2 = 203, respectively). Association with the outcome was tested by Kaplan−Meier, log-rank and multivariate Cox regression analysis in patients with confirmed IDH wild-type status. The biological effects and downstream mechanisms of PLOD2 were assessed in stable PLOD2 knock-down GBM cell lines. High levels of PLOD2 significantly associated with (p1 = 0.020; p2< 0.001; log-rank) and predicted (cohort 1: HR = 1.401, CI [95%] = 1.009−1.946, p1 = 0.044; cohort 2: HR = 1.493; CI [95%] = 1.042−2.140, p2 = 0.029; Cox regression) the poor overall survival of GBM patients. PLOD2 knock-down inhibited tumor proliferation, invasion and anchorage-independent growth. MT1-MMP, CD44, CD99, Catenin D1 and MMP2 were downstream of PLOD2 in GBM cells. GBM cells produced soluble factors via PLOD2, which subsequently induced neutrophils to acquire a pro-tumor phenotype characterized by prolonged survival and the release of MMP9. Importantly, GBM patients with synchronous high levels of PLOD2 and neutrophil infiltration had significantly worse overall survival (p < 0.001; log-rank) compared to the other groups of GBM patients. These findings indicate that PLOD2 promotes GBM progression and might be a useful therapeutic target in this type of cancer.

The Diminishment of Novel Endometrial Carcinoma-Derived Stem-like Cells by Targeting Mitochondrial Bioenergetics and MYC.

Cancer stem cells (CSCs) are a small subpopulation of tumor cells harboring properties that include self-renewal, multi-lineage differentiation, tumor reconstitution, drug resistance and invasiveness, making them key players in tumor relapse. In the present paper, we develop new CSC models and analyze the molecular pathways involved in survival to identify targets for the establishment of novel therapies. Endometrial carcinoma-derived stem-like cells (ECSCs) were isolated from carcinogenic gynecological tissue and analyzed regarding their expression of prominent CSC markers. Further, they were treated with the MYC-signaling inhibitor KJ-Pyr-9, chemotherapeutic agent carboplatin and type II diabetes medication metformin. ECSC populations express common CSC markers, such as Prominin-1 and CD44 antigen as well as epithelial-to-mesenchymal transition markers, Twist, Snail and Slug, and exhibit the ability to form free-floating spheres. The inhibition of MYC signaling and treatment with carboplatin as well as metformin significantly reduced the cell survival of ECSC-like cells. Further, treatment with metformin significantly decreased the mitochondrial membrane potential of ECSC-like cells, while the extracellular lactate concentration was increased. The established ECSC-like populations represent promising in vitro models to further study the contribution of ECSCs to endometrial carcinogenesis. Targeting MYC signaling as well as mitochondrial bioenergetics has shown promising results in the diminishment of ECSCs, although molecular signaling pathways need further investigations.

Primary Sleeve Gastrectomy and Leaks: The Impact of Fundus-Wall Thickness and Staple Heights on Leakage-An Observational Study of 500 Patients.

Introduction: The most feared complication of laparoscopic sleeve gastrectomy (LSG) is staple-line leakage. Staple height and fundus-wall thickness might influence such leakage, and this study examined their possible impact on leak incidence. Factors including gender, age, comorbidities, and reinforcement of the staple line were also investigated. Methods: A total of 500 patients between 17 and 71 years of age who were scheduled for LSG were selected to participate in the study. For technical reasons, 53 were excluded. The fundus-wall thickness of 447 patients after LSG was investigated. The impact of staple height, fundus-wall thickness, demographic and medical factors on leak incidence were investigated. Most of our patients (309) were female (69%), while 138 were male (31%). Results: The mean thickness of the proximal fundus wall was 2,904 µm, 3,172 µm in men and 2,784 µm in women. The leak rate was 4.9%. Age, fundus-wall thickness, and BMI showed a strong influence on leak risk, but this effect was significant only for age (p = 0.01). Patient gender and staple size showed no significant influence on the correlation between fundus-wall thickness and leak risk. Gender displayed a small effect of influence on this correlation, with η2 = 0.05. Discussion: Because older age had a significant effect on increasing the risk of staple-line leakage, there is a need for a more specific focus on these patients. Thinner fundus wall and female gender might predispose patients to staple-line leaks, but a significant value could not be reached. Therefore, staple size should remain the surgeon's choice based on clinical experience.

Analysis of Several Pathways for Efficient Killing of Prostate Cancer Stem Cells: A Central Role of NF-κB RELA.

Prostate cancer is a common cause of death worldwide. Here, we isolated cancer stem cells (CSCs) from four adenocarcinomas of the prostate (Gleason scores from 3 + 3 up to 4 + 5). CSCs were characterized by the expression of the stem cell markers TWIST, the epithelial cell adhesion molecule (EPCAM), the transcription factors SNAI1 (SNAIL) and SNAI2 (SLUG) and cancer markers such as CD44 and prominin-1 (CD133). All investigated CSC populations contained a fraction highly positive for aldehyde dehydrogenase (ALDH) function and displayed robust expressions of programmed cell death 1 (PD-1) ligands. Furthermore, we investigated immunotherapeutic approaches but had no success even with the clinically used PD-1 inhibitor pembrolizumab. In addition, we studied another death-inducing pathway via interferon gamma signaling and detected high-level upregulations of human leukocyte antigen A (HLA-A) and beta 2-microglobulin (B2M) with only moderate killing efficacy. To examine further killing mechanisms in prostate cancer stem cells (PCSCs), we analyzed NF-κB signaling. Surprisingly, two patient-specific populations of PCSCs were found: one with canonical NF-κB signaling and another one with blunted NF-κB activation, which can be efficiently killed by tumor necrosis factor (TNF). Thus, culturing of PCSCs and analysis of respective NF-κB induction potency after surgery might be a powerful tool for optimizing patient-specific treatment options, such as the use of TNF-inducing chemotherapeutics and/or NF-κB inhibitors.

Severe drug-induced liver injury caused by levetiracetam - A case report and review of the literature.

Levetiracetam (LEV) is a broad-spectrum, second-generation anti-seizure medication, which has quickly become one of the most commonly prescribed drugs for people with epielpsy due to its good tolerability, rapid up-dosing capability, with both parenteral and enteral routes of administration. Considering the frequent prescriptions and predominant excretion by the kidney with minimal hepatic metabolism, severe liver injury is very rarely a complication associated with LEV. An analysis of this reported case and further published cases was performed with respect to indication, relevant previous liver diseases, concomitant medication, and both the dosage as well as the duration of LEV when drug-induced liver injury (DILI) was noted. DILI occurs after a few days to a maximum of five months after initiation of therapy with LEV and, in the worst case, may require liver transplantation or result in death. Monitoring of serum transaminase values may be helpful. Discontinuing LEV is the first therapeutic measure. In addition, immunosuppression with cortisone can be considered for serious cases.

Dynein Light Chain Protein Tctex1: A Novel Prognostic Marker and Molecular Mediator in Glioblastoma.

The purpose of this study was to determine the role of Tctex1 (DYNLT1, dynein light chain-1) in the pathophysiology of glioblastoma (GBM). To this end, we performed immunohistochemical analyses on tissues from GBM patients (n = 202). Tctex1 was additionally overexpressed in two different GBM cell lines, which were then evaluated in regard to their proliferative and invasive properties. We found that Tctex1 levels were significantly higher in GBM compared to healthy adjacent brain tissues. Furthermore, high Tctex1 expression was significantly associated with the short overall- (p = 0.002, log-rank) and progression-free (p = 0.028, log-rank) survival of GBM patients and was an independent predictor of poor overall survival in multivariate Cox-regression models. In vitro, Tctex1 promoted the metabolic activity, anchorage-independent growth and proliferation of GBM cells. This phenomenon was previously shown to occur via the phosphorylation of retinoblastoma protein (phospho-RB). Here, we found a direct and significant correlation between the levels of Tctex1 and phospho-RB (Ser807/801) in tissues from GBM patients (p = 0.007, Rho = 0.284, Spearman's rank). Finally, Tctex1 enhanced the invasiveness of GBM cells and the release of pro-invasive matrix metalloprotease 2 (MMP2). These findings indicate that Tctex1 promotes GBM progression and therefore might be a useful therapeutic target in this type of cancer.

Rhinovirus-induced Human Lung Tissue Responses Mimic Chronic Obstructive Pulmonary Disease and Asthma Gene Signatures.

Human rhinovirus (RV) is a major risk factor for chronic obstructive pulmonary disease (COPD) and asthma exacerbations. The exploration of RV pathogenesis has been hampered by a lack of disease-relevant model systems. We performed a detailed characterization of host responses to RV infection in human lung tissue ex vivo and investigated whether these responses are disease relevant for patients with COPD and asthma. In addition, impact of the viral replication inhibitor rupintrivir was evaluated. Human precision-cut lung slices (PCLS) were infected with RV1B with or without rupintrivir. At Days 1 and 3 after infection, RV tissue localization, tissue viability, and viral load were determined. To characterize host responses to infection, mediator and whole genome analyses were performed. RV successfully replicated in PCLS airway epithelial cells and induced both antiviral and proinflammatory cytokines such as IFNα2a, CXCL10, CXCL11, IFN-γ, TNFα, and CCL5. Genomic analyses revealed that RV not only induced antiviral immune responses but also triggered changes in epithelial cell-associated pathways. Strikingly, the RV response in PCLS was reflective of gene expression changes described in patients with COPD and asthma. Although RV-induced host immune responses were abrogated by rupintrivir, RV-triggered epithelial processes were largely refractory to antiviral treatment. Detailed analysis of RV-infected human PCLS and comparison with gene signatures of patients with COPD and asthma revealed that the human RV PCLS model represents disease-relevant biological mechanisms that can be partially inhibited by a well-known antiviral compound and provide an outstanding opportunity to evaluate novel therapeutics.

Novel Primary Human Cancer Stem-Like Cell Populations from Non-Small Cell Lung Cancer: Inhibition of Cell Survival by Targeting NF-κB and MYC Signaling.

There is growing evidence that cancer stem cells (CSCs), a small subpopulation of self-renewal cancer cells, are responsible for tumor growth, treatment resistance, and cancer relapse and are thus of enormous clinical interest. Here, we aimed to isolate new CSC-like cells derived from human primary non-small cell lung cancer (NSCLC) specimens and to analyze the influence of different inhibitors of NF-κB and MYC signaling on cell survival. CSC-like cells were established from three squamous cell carcinomas (SCC) and three adenocarcinomas (AC) of the lung and were shown to express common CSC markers such as Prominin-1, CD44-antigen, and Nestin. Further, cells gave rise to spherical cancer organoids. Inhibition of MYC and NF-κB signaling using KJ-Pyr-9, dexamethasone, and pyrrolidinedithiocarbamate resulted in significant reductions in cell survival for SCC- and AC-derived cells. However, inhibition of the protein-protein interaction of MYC/NMYC proto-oncogenes with Myc-associated factor X (MAX) using KJ-Pyr-9 revealed the most promising survival-decreasing effects. Next to the establishment of six novel in vitro models for studying NSCLC-derived CSC-like populations, the presented investigations might provide new insights into potential novel therapies targeting NF-κB/MYC to improve clinical outcomes in NSCLC patients. Nevertheless, the full picture of downstream signaling still remains elusive.

First case of postmortem study in a patient vaccinated against SARS-CoV-2.

A previously symptomless 86-year-old man received the first dose of the BNT162b2 mRNA COVID-19 vaccine. He died 4 weeks later from acute renal and respiratory failure. Although he did not present with any COVID-19-specific symptoms, he tested positive for SARS-CoV-2 before he died. Spike protein (S1) antigen-binding showed significant levels for immunoglobulin (Ig) G, while nucleocapsid IgG/IgM was not elicited. Acute bronchopneumonia and tubular failure were assigned as the cause of death at autopsy; however, we did not observe any characteristic morphological features of COVID-19. Postmortem molecular mapping by real-time polymerase chain reaction revealed relevant SARS-CoV-2 cycle threshold values in all organs examined (oropharynx, olfactory mucosa, trachea, lungs, heart, kidney and cerebrum) except for the liver and olfactory bulb. These results might suggest that the first vaccination induces immunogenicity but not sterile immunity.

Gene networks and transcriptional regulators associated with liver cancer development and progression.

BACKGROUND: Treatment options for hepatocellular carcinoma (HCC) are limited, and overall survival is poor. Despite the high frequency of this malignoma, its basic disease mechanisms are poorly understood. Therefore, the aim of this study was to use different methodological approaches and combine the results to improve our knowledge on the development and progression of HCC. METHODS: Twenty-three HCC samples were characterized by histological, morphometric and cytogenetic analyses, as well as comparative genomic hybridization (aCGH) and genome-wide gene expression followed by a bioinformatic search for potential transcriptional regulators and master regulatory molecules of gene networks. RESULTS: Histological evaluation revealed low, intermediate and high-grade HCCs, and gene expression analysis split them into two main sets: GE1-HCC and GE2-HCC, with a low and high proliferation gene expression signature, respectively. Array-based comparative genomic hybridization demonstrated a high level of chromosomal instability, with recurrent chromosomal gains of 1q, 6p, 7q, 8q, 11q, 17q, 19p/q and 20q in both HCC groups and losses of 1p, 4q, 6q, 13q and 18q characteristic for GE2-HCC. Gene expression and bioinformatics analyses revealed that different genes and gene regulatory networks underlie the distinct biological features observed in GE1-HCC and GE2-HCC. Besides previously reported dysregulated genes, the current study identified new candidate genes with a putative role in liver cancer, e.g. C1orf35, PAFAH1B3, ZNF219 and others. CONCLUSION: Analysis of our findings, in accordance with the available published data, argues in favour of the notion that the activated E2F1 signalling pathway, which can be responsible for both inappropriate cell proliferation and initial chromosomal instability, plays a pivotal role in HCC development and progression. A dedifferentiation switch that manifests in exaggerated gene expression changes might be due to turning on transcriptional co-regulators with broad impact on gene expression, e.g. POU2F1 (OCT1) and NFY, as a response to accumulating cell stress during malignant development. Our findings point towards the necessity of different approaches for the treatment of HCC forms with low and high proliferation signatures and provide new candidates for developing appropriate HCC therapies.

Hepatic Vasculopathy and Regenerative Responses of the Liver in Fatal Cases of COVID-19.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects the nasopharynx and lungs and causes coronavirus disease-2019 (COVID-19). It may impact the heart, brain, kidney, and liver.1 Although functional impairment of the liver has been correlated with worse clinical outcomes, little is known about the pathophysiology of hepatic injury and repair in COVID-19.2,3 Histologic evaluation has been limited to small numbers of COVID-19 cases with no control subjects2,4 and demonstrated largely heterogeneous patterns of pathology.2,3.

Impact of human papillomaviruses (HPV) on recurrence rate and malignant progression of sinonasal papillomas.

Sinonasal papillomas are characterized by their potential for frequent recurrences and malignant progression. Currently, the role of human papillomavirus (HPV) infection in sinonasal papillomas is unclear. A study was conducted to elucidate the impact of HPV infection on recurrence and malignant progression of sinonasal papillomas. One hundred and seven patients with 151 tumors could be examined. One hundred and one patients suffered from benign papilloma, mostly inverted papillomas (IP); six patients suffered from carcinomas in situ and squamous cell carcinomas (SCC) ex-IP. Recurrent IP were more often HPV-positive than non-recurrent tumors (38.8% vs. 60%-65%). Low-risk (LR) HPV infection (especially HPV 6) increased the risk of tumor recurrences (p = 0.0385 and p = 0.0556, respectively). IP and oncocytic papillomas (both lesions are known for their malignant potential) were more often high-risk (HR) HPV-positive (15.5% and 16.7%) than fungiform papilloma (which usually does not progress to carcinoma). CIS and SCC ex-IP displayed higher HPV rates than benign IP (83.3% vs. 38.8%), especially higher rates of HR-HPV (66.7% vs. 23.8%, p = 0.0415). Data from this study endorse the hypothesis that recurrence of sinonasal papillomas is promoted by LR-HPV infection and that malignant progression of IP is promoted by HR-HPV infection.

Targeting interleukin 6 signaling by monoclonal antibody siltuximab on cholangiocarcinoma.

BACKGROUND AND AIM: Cholangiocarcinoma has an unimproved prognosis. Interleukin 6 (IL-6) has an oncogenic potential in some cancer diseases. However, the role of IL-6 in cholangiocarcinoma carcinogenesis is not well understood. The current study investigated the role of IL-6 signaling in cholangiocarcinoma carcinogenesis and efficacy of siltuximab treatment on cholangiocarcinoma in vitro and in vivo. METHODS: The expression of IL-6 was analyzed on human cholangiocarcinoma cell lines and murine and human cholangiocarcinoma tissues, using reverse transcription real-time polymerase chain reaction and immunohistochemistry. In addition, the effect of anti-IL-6 chimeric monoclonal antibody, siltuximab, was investigated in vitro by proliferation, migration, and two-dimensional and three-dimensional invasion assays and in vivo by xenograft mouse model. Western blot was applied to study the molecular alteration. RESULTS: Our result shows high expression of IL-6 in human cholangiocarcinoma cells, and IL-6 stimulants enhance cholangiocarcinoma cell proliferation. In addition, murine and human cholangiocarcinoma tissues express significantly higher levels of IL-6, compared with adjacent non-tumor tissues. On the cholangiocarcinoma engineered mouse model, IL-6 level is associated with tumor volume. Taken together, our data indicate an oncogenic potential of IL-6 in cholangiocarcinoma carcinogenesis. Siltuximab sufficiently abrogates IL-6 signaling and inhibits cholangiocarcinoma progression in vitro and in vivo. The results additionally indicate a relative alteration of IL-6 signaling and its molecular targets, such as STAT3, Wnt/ß-catenin, and mesenchymal markers. CONCLUSIONS: Interleukin 6 plays an essential role in cholangiocarcinoma carcinogenesis, and siltuximab has the potential to be considered as a new treatment option for cholangiocarcinoma patients.

TMEM16A/ANO1 calcium-activated chloride channel as a novel target for the treatment of human respiratory syncytial virus infection.

INTRODUCTION: Human respiratory syncytial virus (HRSV) is a common cause of respiratory tract infections (RTIs) globally and is one of the most fatal infectious diseases for infants in developing countries. Of those infected, 25%-40% aged ≤1 year develop severe lower RTIs leading to pneumonia and bronchiolitis, with ~10% requiring hospitalisation. Evidence also suggests that HRSV infection early in life is a major cause of adult asthma. There is no HRSV vaccine, and the only clinically approved treatment is immunoprophylaxis that is expensive and only moderately effective. New anti-HRSV therapeutic strategies are therefore urgently required. METHODS: It is now established that viruses require cellular ion channel functionality to infect cells. Here, we infected human lung epithelial cell lines and ex vivo human lung slices with HRSV in the presence of a defined panel of chloride (Cl-) channel modulators to investigate their role during the HRSV life-cycle. RESULTS: We demonstrate the requirement for TMEM16A, a calcium-activated Cl- channel, for HRSV infection. Time-of-addition assays revealed that the TMEM16A blockers inhibit HRSV at a postentry stage of the virus life-cycle, showing activity as a postexposure prophylaxis. Another important negative-sense RNA respiratory pathogen influenza virus was also inhibited by the TMEM16A-specific inhibitor T16Ainh-A01. DISCUSSION: These findings reveal TMEM16A as an exciting target for future host-directed antiviral therapeutics.

Impact of ASA-score, age and learning curve on early outcome in the initiation phase of an oncological robotic colorectal program.

The ASA score is known to be an independent predictor of complications and mortality following colorectal surgery. We evaluated early outcome in the initiation phase of a robotic oncological colorectal resection program in dependence of comorbidity and learning curve. 43 consecutive colorectal cancer patients (median age: 74 years) who underwent robotic surgery were firstly analysed defined by physical status (group A = ASA1 + 2; group B = ASA3). Secondly, outcome was evaluated relating to surgery date (group E: early phase; group L: late phase). There were no differences among groups A and B with regard to gender, BMI, skin-to-skin operative times (STS), N- and M-status, hospital-stay as well as overall rate of complications according to Dindo-Clavien and no one-year mortality. GroupA when compared to group B demonstrated significantly lower mean age (65.5 years ± 11.4 years vs 75.8 years ± 8.9 years), T-stage and ICU-stay. When separately analyzed for patients age ICU-stay was comparable (> 75 years vs. < 75 years). Group E and L demonstrated comparable characteristics and early outcome except more frequent lymphatic fistulas in group E. STS was reduced in group L compared to group E. Beyond learning curve aspects in our series, we could demonstrate that patient's physical condition according to ASA rather than age may have an impact on early outcome in the initial phase of a robotic oncological colorectal program.

Isolation and Characterization of Two Novel Colorectal Cancer Cell Lines, Containing a Subpopulation with Potential Stem-Like Properties: Treatment Options by MYC/NMYC Inhibition.

Cancer stem cells (CSC) are crucial mediators of cancer relapse. Here, we isolated two primary human colorectal cancer cell lines derived from a rectal neuroendocrine carcinoma (BKZ-2) and a colorectal adenocarcinoma (BKZ-3), both containing subpopulations with potential stem-like properties. Protein expression of CSC-markers prominin-1 and CD44 antigen was significantly higher for BKZ-2 and BKZ-3 in comparison to well-established colon carcinoma cell lines. High sphere-formation capacity further confirmed the existence of a subpopulation with potential stem-like phenotype. Epithelial-mesenchymal transition markers as well as immune checkpoint ligands were expressed more pronounced in BKZ-2. Both cell populations demonstrated N-myc proto-oncogene (NMYC) copy number gain. Myc proto-oncogene (MYC)/NMYC activity inhibitor all-trans retinoic acid (ATRA) significantly reduced the number of tumor spheres for both and the volume of BKZ-2 spheres. In contrast, the sphere volume of ATRA-treated BKZ-3 was increased, and only BKZ-2 cell proliferation was reduced in monolayer culture. Treatment with KJ-Pyr-9, a specific inhibitor of MYC/NMYC-myc-associated factor X interaction, decreased survival by the induction of apoptosis of both. In summary, here, we present the novel colorectal cancer cell lines BKZ-2 and BKZ-3 as promising cellular in vitro models for colorectal carcinomas and identify the MYC/NMYC molecular pathway involved in CSC-induced carcinogenesis with relevant therapeutic potential.

Haprolid Inhibits Tumor Growth of Hepatocellular Carcinoma through Rb/E2F and Akt/mTOR Inhibition.

BACKGROUND: Hepatocellular carcinoma (HCC) represents a major health burden with limited curative treatment options. There is a substantial unmet need to develop innovative approaches to impact the progression of advanced HCC. Haprolid is a novel natural component isolated from myxobacteria. Haprolid has been reported as a potent selective cytotoxin against a panel of tumor cells in recent studies including HCC cells. The aims of this study are to evaluate the antitumor effect of haprolid in HCC and to understand its underlying molecular mechanisms. METHODS: The efficacy of haprolid was evaluated in human HCC cell lines (Huh-7, Hep3B and HepG2) and xenograft tumors (NMRI-Foxn1nu mice with injection of Hep3B cells). Cytotoxic activity of haprolid was determined by the WST-1 and crystal violet assay. Wound healing, transwell and tumorsphere assays were performed to investigate migration and invasion of HCC cells. Apoptosis and cell-cycle distribution were measured by flow cytometry. The effects of haprolid on the Rb/E2F and Akt/mTOR pathway were examined by immunoblotting and immunohistochemistry. RESULTS: haprolid treatment significantly inhibited cell proliferation, migration and invasion in vitro. The epithelial-mesenchymal transition (EMT) was impaired by haprolid treatment and the expression level of N-cadherin, vimentin and Snail was downregulated. Moreover, growth of HCC cells in vitro was suppressed by inhibition of G1/S transition, and partially by induction of apoptosis. The drug induced downregulation of cell cycle regulatory proteins cyclin A, cyclin B and CDK2 and induced upregulation of p21 and p27. Further evidence showed that these effects of haprolid were associated with Rb/E2F downregulation and Akt/mTOR inhibition. Finally, in vivo nude mice experiments demonstrated significant inhibition of tumor growth upon haprolid treatment. CONCLUSION: Our results show that haprolid inhibits the growth of HCC through dual inhibition of Rb/E2F and Akt/mTOR pathways. Therefore, haprolid might be considered as a new and promising candidate for the palliative therapy of HCC.