Eradication of chemotherapy-resistant CD44+ human ovarian cancer stem cells in mice by intraperitoneal administration of Clostridium perfringens enterotoxin

BACKGROUND:

Emerging evidence has suggested that the capability to sustain tumor formation, growth, and chemotherapy resistance in ovarian as well as other human malignancies exclusively resides in a small proportion of tumor cells termed cancer stem cells. During the characterization of CD44⁺ ovarian cancer stem cells, we found a high expression of the genes encoding for claudin‐4. Because this tight junction protein is the natural high‐affinity receptor for Clostridium perfringens enterotoxin (CPE), we have extensively investigated the sensitivity of ovarian cancer stem cells to CPE treatment in vitro and in vivo.

METHODS:

Real‐time polymerase chain reaction and flow cytometry were used to evaluate claudin‐3/‐4 expression in ovarian cancer stem cells. Small interfering RNA knockdown experiments and MTS assays were used to evaluate CPE‐induced cytotoxicity against ovarian cancer stem cell lines in vitro. C.B‐17/SCID mice harboring ovarian cancer stem cell xenografts were used to evaluate CPE therapeutic activity in vivo.

RESULTS:

CD44⁺ ovarian cancer stem cells expressed claudin‐4 gene at significantly higher levels than matched autologous CD44^? ovarian cancer cells, and regardless of their higher resistance to chemotherapeutic agents died within 1 hour after exposure to 1.0 μg/mL of CPE in vitro. Conversely, small‐interfering RNA‐mediated knockdown of claudin‐3/‐4 expression in CD44⁺ cancer stem cells significantly protected cancer stem cells from CPE‐induced cytotoxicity. Importantly, multiple intraperitoneal administrations of sublethal doses of CPE in mice harboring xenografts of chemotherapy‐resistant CD44⁺ ovarian cancer stem cells had a significant inhibitory effect on tumor progression leading to the cure and/or long‐term survival of all treated animals (ie, 100% reduction in tumor burden in 50% of treated mice; P < .0001).

CONCLUSIONS:

CPE may represent an unconventional, potentially highly effective strategy to eradicate chemotherapy‐resistant cancer stem cells. Cancer 2011;. © 2011 American Cancer Society.     

Gene expression fingerprint of uterine serous papillary carcinoma: identification of novel molecular markers for uterine serous cancer diagnosis and therapy

Uterine serous papillary cancer (USPC) represents a rare but highly aggressive variant of endometrial cancer, the most common gynecologic tumour in women. We used oligonucleotide microarrays that interrogate the expression of some 10 000 known genes to profile 10 highly purified primary USPC cultures and five normal endometrial cells (NEC). We report that unsupervised analysis of mRNA fingerprints readily distinguished USPC from normal endometrial epithelial cells and identified 139 and 390 genes that exhibited >5-fold upregulation and downregulation, respectively, in primary USPC when compared to NEC. Many of the genes upregulated in USPC were found to represent adhesion molecules, secreted proteins and oncogenes, such as L1 cell adhesion molecule, claudin-3 and claudin-4, kallikrein 6 (protease M) and kallikrein 10 (NES1), interleukin-6 and c-erbB2. Downregulated genes in USPC included SEMACAP3, ras homolog gene family, member I (ARHI), and differentially downregulated in ovarian carcinoma gene 1. Quantitative RT-PCR was used to validate differences in gene expression between USPC and NEC for several of these genes. Owing to its potential as a novel therapeutic marker, expression of the high-affinity epithelial receptor for Clostridium perfringens enterotoxin (CPE) claudin-4 was further validated through immunohistochemical analysis of formalin-fixed paraffin-embedded specimens from which the primary USPC cultures were obtained, as well as an independent set of archival USPC specimens. Finally, the sensitivity of primary USPC to the administration of scalar doses of CPE in vitro was also demonstrated. Our results highlight the novel molecular features of USPC and provide a foundation for the development of new type-specific therapies against this highly aggressive variant of endometrial cancer.     

Serum amyloid A (SAA): a novel biomarker for uterine serous papillary cancer

Background:

Uterine serous papillary carcinoma (USPC) is a biologically aggressive variant of endometrial cancer. We investigated the expression of Serum Amyloid A (SAA) and evaluated its potential as a serum biomarker in USPC patients.

Methods:

SAA gene and protein expression levels were evaluated in USPC and normal endometrial tissues (NEC) by real-time PCR, immunohistochemistry (IHC), flow cytometry and by a sensitive bead-based immunoassay. SAA concentration in 123 serum samples from 51 healthy women, 42 women with benign diseases, and 30 USPC patients were also studied.

Results:

SAA gene expression levels were significantly higher in USPC when compared with NEC (mean copy number by RT–PCR=162 vs 2.21; P=0.0002). IHC revealed diffuse cytoplasmic SAA protein staining in USPC tissues. High intracellular levels of SAA were identified in primary USPC cell lines evaluated by flow cytometry and SAA was found to be actively secreted in vitro. SAA concentrations (μg ml⁻¹) had a median (95% CIs) of 6.0 (4.0–8.9) in normal healthy females and 6.0 (4.2–8.1) in patients with benign disease (P=0.92). In contrast, SAA values in the serum of USPC patients had a median (95% CI) of 15.6 (9.2–56.2), significantly higher than those in the healthy group (P=0.0005) and benign group (P=0.0006). Receiver operating characteristics (ROC) analysis of serum SAA to classify advanced- and early-stage USPC yielded an area under the ROC curve of 0.837 (P=0.0024).

Conclusion:

SAA is not only a liver-secreted protein but is also a USPC cell product. SAA may represent a novel biomarker for USPC to assist in staging patients preoperatively, and to monitor early-disease recurrence and response to therapy.     

Uterine serous papillary carcinomas overexpress human trophoblast-cell-surface marker (Trop-2) and are highly sensitive to immunotherapy with hRS7, a humanized anti-Trop-2 monoclonal antibody

BACKGROUND:

Uterine serous papillary carcinoma (USPC) was an aggressive and chemotherapy resistant variant of endometrial cancer. The authors evaluated the expression of human trophoblast‐cell‐surface‐marker (Trop‐2) and the potential of hRS7, a humanized anti‐Trop‐2 monoclonal antibody, as a novel therapeutic strategy against USPC.

METHODS:

Trop‐2 expression was evaluated by immunohistochemistry (IHC) in a total of 23 USPC. Six primary USPC cell lines were assessed by flow cytometry and real‐time polymerase chain reaction (PCR) for Trop‐2 expression. Sensitivity to hRS7 (Immunomedics, Inc.) antibody‐dependent cellular cytotoxicity (ADCC) and complement‐dependent cytotoxicity was tested in standard 5‐hour ⁵¹Cr‐release assays against primary USPC cell lines.

RESULTS:

Expression of Trop‐2 was found in 15 of 23 (65%) of the tumor tissues tested by IHC and in 50% (3 of 6) of the USPC cell lines tested by real‐time PCR and flow‐cytometry (Trop‐2 expression in USPC versus normal endometrial cells; P < .005). USPC cell lines overexpressing Trop‐2, regardless of their intrinsic resistance to natural killer cytotoxicity, were highly sensitive to hRS7‐mediated ADCC in vitro (range of killing, 28.2% to 64.4%) (P < .001). Negligible cytotoxicity against USPC was seen in the absence of hRS7 or in the presence of rituximab control antibody (range of killing, 1.1% to 12.4%). Incubation with interleukin‐2 (50 IU/mL) in addition to hRS7 further increased the cytotoxic activity against USPC cell lines overexpressing Trop‐2 (P = .008).

CONCLUSIONS:

Trop‐2 was highly expressed in uterine serous carcinoma at mRNA and protein levels. Primary USPC cell lines are highly sensitivity to hRS7‐mediated cytotoxicity in vitro. hRS7 may represent a novel therapeutic agent for USPC refractory to standard treatment modalities. Cancer 2011. © 2011 American Cancer Society.     

Up-regulation of a novel mRNA (NY-CO-1) involved in the methyl 4-methoxy-3-(3-methyl-2-butenoyl) benzoate (VT1)-induced proliferation arrest of a non-small-cell lung carcinoma cell line (NSCLC-N6)

It is now well known that treatment of tumors, especially non-small-cell lung cancer (NSCLC), remains limited and it is urgent to develop strategies that target tumor cells and their genetic features. In this regard, our work is about genetic modifications arising in an in vitro NSCLC cell line after treatment with a chemical substance, methyl 4-methoxy-3-(3-methyl-2-butenoyl) benzoate (VT1). First, we showed that VT1 induces arrest of proliferation by blocking cells in the G1 phase of the cell cycle. Second, we use "differential display" strategy to clarify the genetic mechanisms involved in this proliferation arrest. A novel mRNA, NY-CO-1 (New-York Colon 1), of unknown function showed up-regulated expression after treatment. Application of "antisense" strategy confirmed this novel mRNA induction was effectively linked to growth arrest. Therefore, these data provide new information about mechanisms participating in arrest of proliferation of tumor cells and open new ways of treatment to target tumor growth.     

Identification of cytotoxic T-lymphocyte epitope(s) and its agonist epitope(s) of a novel target for vaccine therapy (PAGE4)

PAGE4 is an X chromosome-linked cancer testis antigen and is a potential new tumor-associated antigen that is overexpressed in prostate and uterine cancers. The purpose of this study was to identify a human CTL epitope and a corresponding agonist epitope of PAGE4 to determine if PAGE4 is a potential target for vaccine-mediated immunotherapy against PAGE4-expressing tumors. A class I PAGE4 epitope was identified with a high level of binding to HLA-A2. PAGE4 peptide-pulsed dendritic cells were then used to generate human PAGE4-specific T-cell lines. Further studies demonstrated the generation of an enhancer agonist epitope. Compared with the native peptide, the agonist (i) bound to HLA-A2 molecules at lower peptide concentrations, (ii) demonstrated a higher stability of the peptide HLA-A2 complex, (iii) induced higher levels of production of IFN-gamma, Granzyme B, TNF-alpha, IL-2 and lymphotactin by PAGE4-specific T-cell lines and (iv) T-cell lines generated against the agonist peptide were more efficient to lyse HLA-A2 human tumor cells expressing native PAGE4. The studies reported here are the first to describe a PAGE4 CTL epitope and its agonist epitope, and thus identify PAGE4 as a potentially useful target for vaccine-mediated therapy of prostate cancer.     

Targeting cytotoxic T-lymphocyte antigen-4 (CTLA-4): a novel strategy for the treatment of melanoma and other malignancies

Cancer immunotherapy centers on modulating the host's tumor-directed immune response. One promising approach involves augmentation of cell-mediated immunity by interrupting T-cell pathways responsible for immune down-regulation or tolerance. The discovery of cytotoxic T-lymphocyte antigen-4 (CTLA-4) and its role as a key negative regulator for T cells has prompted efforts to target this signaling molecule to improve cancer therapy. Activation, or 'priming', of naive T cells in response to tumor-cell invasion comprises a dual-signaling mechanism. Signal 1 requires tumor-associated antigen recognition by the T-cell receptor, while signal 2 occurs through binding of CD80 or CD86 (B7.1 of 2) on the antigen presenting cell (APC) with CD28 on the T cell. Importantly, there is a final step responsible for naturally occurring immune regulation; this occurs in response to competitive binding of CD80/CD86 on the APC by CTLA-4 on the T cell. This 'immune checkpoint' interrupts signal 2 and inhibits the activated T cell. Targeting CTLA-4 as an anticancer strategy: Following proof-of-concept studies in animals, fully human anti-CTLA-4 antibodies were developed and 2 are undergoing clinical evaluation. Ipilimumab and tremelimumab have shown promising antitumor activity, initially in patients with advanced melanoma. Class-specific immune-related adverse events (irAEs) were common and mostly transient and/or manageable. These events are thought to be mechanism-of-action-related, indicating immune tolerance is broken; this relation may also explain the association between irAEs and response seen in several trials. Interruption of immune inhibitory pathways via CTLA-4 blockade appears to be a promising strategy for cancer immunotherapy.     

主动呼吸控制技术用于原发性肝癌放疗的可行性及剂量学研究

目的研究主动呼吸控制技术(ABC)用于原发性肝癌放疗的可行性并且与自由呼吸(FB)的放疗计划进行剂量学参数比较。方法入选病人完成ABC呼吸训练后,进行CT模拟定位,TPS计划设计,摆位验证以及实施放疗,评估ABC应用于原发性肝癌放疗的可行性,ABC放疗的摆位误差和肝脏位置的重复性,并且与平静呼吸的放疗计划比较计划靶体积(PTV)、未受肝癌累及的正常肝脏的平均剂量(MDTNL)、接受≥23Gy照射的肝脏体积比(V23)和放射性肝病的发生概率(NTCP)等剂量学参数。结果有11例病人入选该研究,所有病人配合良好,没有因为不能耐受屏气而中断放疗者。PTV的平均体积由FB下的757cm3±475cm3减少到了ABC技术的444cm3±297cm3(P=0.002)。ABC技术下的平均MDTNL为15.9Gy±5.4Gy,而FB则为24.6Gy±6.5Gy(P<0.01)。ABC计划和FB计划平均的V23分别为31%和55%,而NTCP分别为11%和21%。ABC放疗的随机误差和系统误差在头脚、前后和左右方向分别为4.8mm和1.3mm、3.5mm和1.6mm以及2.4mm和1.7mm,肝脏位置一次放疗内和分次放疗间在头脚方向上的重复性平均分别为1.5mm(范围:0.5～3.0mm)和5.1mm(范围:1.9～9.2mm)。ABC放疗所需的时间平均为6分钟(范围:5～14分钟)。结论ABC技术用于肝癌放疗是可行的。没有显著增加治疗时间,摆位精确性和重复性好。该技术能减少正常肝的照射体积,降低平均剂量,减少了放射性肝病的发生概率。     

Initiation of esophageal squamous cell carcinoma (ESCC) in a murine 4-nitroquinoline-1-oxide and alcohol carcinogenesis model

Esophageal squamous cell carcinomas (ESCCs) are very common, aggressive tumors, and are often associated with alcohol and tobacco abuse. Because ESCCs exhibit high recurrence rates and are diagnosed at late stages, identification of prognostic and drug targets for prevention and treatment is critical. We used the 4-nitroquinoline-1-oxide (4-NQO) murine model of oral carcinogenesis and the Meadows-Cook model of alcohol abuse to assess changes in the expression of molecular markers during the initial stages of ESCC. Combining these two models, which mimic chronic alcohol and tobacco abuse in humans, we detected increased cellular proliferation (EGFR and Ki67 expression), increased canonical Wnt signaling and downstream elements (β-catenin, FoxM1, and S100a4 protein levels), changes in cellular adhesive properties (reduced E-cadherin in the basal layer of the esophageal epithelium), and increased levels of phosphorylated ERK1/2 and p38. Additionally, we found that treatment with ethanol alone increased the numbers of epithelial cells expressing solute carrier family 2 (facilitated glucose transporter, member 1) (SLC2A1) and carbonic anhydrase IX (CAIX), and increased the phosphorylation of p38. Thus, we identified both 4-NQO- and ethanol-specific targets in the initial stages of esophageal carcinogenesis, which should lead to the development of potential markers and therapeutic targets for human ESCC.     

Local recurrence in breast carcinoma patients with T(1-2) and 1-3 positive nodes: indications for radiotherapy.

AIMS: To investigate the relationship between local recurrence (LR) and distant recurrence (DR) and to determine a subgroup of patients who could benefit from radiotherapy among breast carcinoma patients with T(1-2) and N(1a). METHODS: Univariate and multivariate Cox regression analyses were carried out in the retrospective data of 326 eligible patients. RESULTS: Fourteen (4.3%) patients had LR and 46 (14.1%) patients suffered DR, in their follow-up periods. The multivariate time-dependent Cox model for DR showed that ratio of positive nodes (PN) (p=0.004; hazard ratio (HR), 1.05; 95% confidence interval (CI), 1.02-1.09) and LR (p=0.05; HR, dependent on time) were strongly associated with DR. In the multivariate Cox analysis for LR, age (35 years; p<0.0001; HR, 6.8; CI, 2.3-19.9), lymphatic vascular invasion (LVI) (yes vs no; p=0.03; HR, 3.3; CI, 1.2-9.8), and a ratio of PN (>15% vs 相似文献   

Performance of a new HPV and biomarker assay in the management of hrHPV positive women: Subanalysis of the ongoing multicenter TRACE clinical trial (n > 6,000) to evaluate POU4F3 methylation as a potential biomarker of cervical precancer and cancer

The ongoing Triage and Risk Assessment of Cervical Precancer by Epigenetic Biomarker (TRACE) prospective, multicenter study aimed to provide a clinical evaluation of the CONFIDENCE? assay, which comprises a human papillomavirus (HPV) DNA and a human epigenetic biomarker test. Between 2013 and 2015 over 6,000 women aged 18 or older were recruited in Hungary. Liquid‐based cytology (LBC), high‐risk HPV (hrHPV) DNA detection and single target host gene methylation test of the promoter sequence of the POU4F3 gene by quantitative methylation‐specific polymerase chain reaction (PCR) were performed from the same liquid‐based cytology sample. The current analysis is focused on the baseline cross‐sectional clinical results of 5,384 LBC samples collected from subjects aged 25 years or older. The performance of the CONFIDENCE HPV? test was found to be comparable to the cobas® HPV test with good agreement. When applying the CONFIDENCE Marker? test alone in hrHPV positives, it showed significantly higher sensitivity with matching specificity compared to LBC‐based triage. For CIN3+ histological endpoint in the age group of 25–65 and 30–65, the methylation test of POU4F3 achieved relative sensitivities of 1.74 (95% CI: 1.25–2.33) and 1.64 (95% CI: 1.08–2.27), respectively, after verification bias adjustment. On the basis of our findings, POU4F3 methylation as a triage test of hrHPV positives appears to be a noteworthy method. We can reasonably assume that its quantitative nature offers the potential for a more objective and discriminative risk assessment tool in the prevention and diagnostics of high‐grade cervical intraepithelial neoplasia (CIN) lesions and cervical cancer.