A pilot study of multi-antigen stimulated cell therapy-I plus camrelizumab and apatinib in patients with advanced bone and soft-tissue sarcomas.

BACKGROUND: Cell-based  immunotherapy shows the therapeutic potential in sarcomas, in addition to angiogenesis-targeted tyrosine kinase inhibitor (TKI) and immune checkpoint inhibitor (ICI). Multi-antigen stimulated cell therapy-I (MASCT-I) technology is a sequential immune cell therapy for cancer, which composes of multiple antigen-loaded dendritic cell (DC) vaccines followed by the adoptive transfer of anti-tumor effector T-cells. METHODS: In this phase 1 study, we assessed MASCT-I plus camrelizumab (an ICI against PD-1) and apatinib (a highly selective TKI targeting VEGFR2) in patients with unresectable recurrent or metastatic bone and soft-tissue sarcoma after at least one line of prior systemic therapy. One MASCT-I course consisted of 3 DC subcutaneous injections, followed by 3 active T cell infusions administered 18-27 days after each DC injection. In schedule-I group, 3 DC injections were administered with a 28-day interval in all courses; in schedule-II group, 3 DC injections were administered with a 7-day interval in the first course and with a 28-day interval thereafter. All patients received intravenous camrelizumab 200 mg every 3 weeks and oral apatinib 250 mg daily. RESULTS: From October 30, 2019, to August 12, 2021, 19 patients were enrolled and randomly assigned to schedule-I group (n = 9) and schedule-II group (n = 10). Of the 19 patients, 11 (57.9%) experienced grade 3 or 4 treatment-related adverse events. No treatment-related deaths occurred. Patients in schedule-II group showed similar objective response rate (ORR) with those in schedule-I group (30.0% versus 33.3%) but had higher disease control rate (DCR; 90.0% versus 44.4%) and longer median progression-free survival (PFS; 7.7 versus 4.0 months). For the 13 patients with soft-tissue sarcomas, the ORR was 30.8%, DCR was 76.9%, and median PFS was 12.9 months; for the 6 patients with osteosarcomas, the ORR was 33.3%, the DCR was 50.0%, and median PFS was 5.7 months. CONCLUSIONS: Overall, MASCT-I plus camrelizumab and apatinib was safe and showed encouraging efficacy in advanced bone and soft-tissue sarcoma, and schedule-II administration method was recommended. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04074564.

Impact of Secondary Aneurysmal Bone Cysts on Survival of Patients with Enneking Stage IIB Extremity Osteosarcoma: A Propensity Score Matching Analysis.

BACKGROUND: The clinicopathology of aneurysmal bone cysts (ABCs) secondary to osteosarcoma has not yet been reported. We conduct a retrospective review of ABCs secondary to osteosarcoma to characterize clinicopathology and influence on the survival of patients with Enneking stage IIB extremity osteosarcoma. PATIENTS AND METHODS: A total of 300 patients with Enneking stage IIB extremity osteosarcoma were eligible for analysis. These cases were divided, according to the pathology of biopsy and magnetic resonance imaging (MRI), into ABCs group and no ABCs group. Patients (ABCs versus no ABCs) were compared using a 1:2 propensity score analysis to best match between groups. Clinicopathology and survival data were analyzed. RESULTS: The total occurrence rate of secondary ABCs was 10.3%. A higher prevalence of pathological fractures was observed in the ABCs group (22.6%) compared with the no ABCs group (8.6%) (p = 0.032). Patients with ABCs were more likely to undergo amputation compared with patients without ABCs (p = 0.007). Those with secondary ABCs had poorer response to chemotherapy before and after propensity score matching (p = 0.006 and p = 0.048, respectively). Kaplan-Meier survival analysis showed that EFS and OS distributions were not significantly different between the two patient groups. ABCs were not significantly different in terms of EFS or OS in the multivariate analysis model (p > 0.05). CONCLUSIONS: The presence of secondary ABCs was associated with increased occurrence rate of pathological fracture and high percentage of amputation. Moreover, patients with secondary ABCs had poorer response to chemotherapy. However, the presence of secondary ABCs did not influence survival of patients with Enneking stage IIB extremity osteosarcoma.

Clinical significance of indeterminate pulmonary nodules on the survival of 364 patients with nonmetastatic, high-grade, localized osteosarcoma: A 12-year retrospective cohort study.

BACKGROUND AND OBJECTIVES: We investigated the clinical significance of indeterminate pulmonary nodules (IPNs) in patients diagnosed with nonmetastatic, high-grade localized osteosarcoma. METHODS: We retrospectively analyzed the clinical data of 364 patients with nonmetastatic, high-grade localized osteosarcoma. Based on pulmonary computed tomography findings at presentation, the patients were categorized into the no-nodules and the IPNs group and were further categorized into subgroups based on age (<18 and ≥18 years). We performed an intergroup comparison of event-free survival (EFS) and overall survival (OS). RESULTS: At presentation, 276 (75.8%) patients showed no nodules, and 88 (24.2%) patients showed IPNs. The EFS and OS were similar between adults with IPNs (n = 54 [30.5%]) and without nodules (n = 123 [69.5%]) (p = .200 and p = .609, respectively). No significant intergroup difference in OS was observed in pediatric patients (p = .093). However, pediatric patients with IPNs (n = 34 [18.2%]) had poorer EFS than those without nodules (n = 153 [81.8%]) (p = .016). Multivariate analyses confirmed that IPNs were independently associated with poorer EFS in pediatric patients (hazard ratio 1.788, 95% confidence interval 1.092-2.926, p = .021). CONCLUSIONS: This study showed that IPNs at presentation did not affect the survival of adults with nonmetastatic, high-grade localized osteosarcoma but were associated with poorer EFS in pediatric patients.

Alterations in Serum Lipids and Lipoproteins Induced by Neoadjuvant Chemotherapy in Patients with Osteosarcoma around the Knee Joint: A Retrospective Analysis.

OBJECTIVE: To investigate the serum lipid profiles of patients with localized osteosarcoma around the knee joint before and after neoadjuvant chemotherapy. METHODS: After retrospectively screening the data of 742 patients between January 2007 and July 2020, 50 patients aged 13 to 39 years with Enneking stage II disease were included in the study. Serum lipid levels, including total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), lipoprotein-α [Lp(a)], and apolipoprotein A1, B, and E (ApoA1, ApoB, and ApoE), and clinicopathological characteristics were collected before and after neoadjuvant chemotherapy. RESULTS: The mean levels of TC, TG, and ApoB were significantly increased following neoadjuvant chemotherapy (16%, 38%, and 20%, respectively, vs. pretreatment values; P<0.01). The mean levels of LDL-C and ApoE were also 19% and 16% higher, respectively (P<0.05). No correlation was found between the pretreatment lipid profile and the histologic response to chemotherapy. An increase in Lp(a) was strongly correlated with the Ki-67 index (R=0.31, P=0.023). Moreover, a trend toward longer disease-free survival (DFS) was observed in patients with decreased TG and increased LDL-C following chemotherapy, although this difference was not statistically significant (P=0.23 and P=0.24, respectively). CONCLUSION: Significant elevations in serum lipids were observed after neoadjuvant chemotherapy in patients with localized osteosarcoma. There was no prognostic significance of pretreatment serum lipid levels on histologic response to neoadjuvant chemotherapy. The scale of increase in serum Lp(a) might have a potential prognostic role in osteosarcoma. Patients with increased LDL-C or reduced TG after chemotherapy seem to exhibit a trend toward favorable DFS.

Standardization of organoid culture in cancer research.

Establishing a valid in vitro model to represent tumor heterogeneity and biology is critical but challenging. Tumor organoids are self-assembled three-dimensional cell clusters which are of great significance for recapitulating the histopathological, genetic, and phenotypic characteristics of primary tissues. The organoid has emerged as an attractive in vitro platform for tumor biology research and high-throughput drug screening in cancer medicine. Organoids offer unique advantages over cell lines and patient-derived xenograft models, but there are no standardized methods to guide the culture of organoids, leading to confusion in organoid studies that may affect accurate judgments of tumor biology. This review summarizes the shortcomings of current organoid culture methods, presents the latest research findings on organoid standardization, and proposes an outlook for organoid modeling.

First-in-Maintenance Therapy for Localized High-Grade Osteosarcoma: An Open-Label Phase I/II Trial of the Anti-PD-L1 Antibody ZKAB001.

PURPOSE: We investigated the safety and preliminary efficacy of anti-PD-L1 antibody (ZKAB001) as maintenance therapy for localized patients with high-grade osteosarcoma to reduce the risk of recurrence and metastasis. PATIENTS AND METHODS: This open-label Phase I/II study was divided into dose-escalation Phase I and expansion Phase II. Phase I used a 3+3 design with ZKAB001 at three escalating doses ranging: 5, 10, 15 mg/kg every 2 weeks in 9 patients with localized high-grade osteosarcoma and Phase II tested 10 mg/kg in 12 patients for up to 24 cycles. Primary endpoints were safety and tolerability assessed using CTCAE4.0.3. RESULTS: Between October 2018 and 2019, 21 eligible patients were enrolled and accepted ZKAB001 treatment: 9 in the dose-escalation phase, and 12 in expansion phase. Six patients with disease progression withdrew from this study and follow-up is ongoing. The MTD was not defined in Phase I. All doses had a manageable safety profile. The recommended dose in Phase II was set at 10 mg/kg. Most frequent immune-related adverse events were thyroiditis (76.2%) and dermatitis (42.9%). Only 1 (4.8%) of 21 patients had a Grade 3 skin rash. The median 3-year event-free survival (EFS) and overall survival (OS) were not established; however, 24-month EFS was 71.4% (95% confidence interval, 47.2-86.0) and 2-year OS was 100%. Preliminary efficacy data showed EFS benefits in patients with PD-L1 positive or an MSI-H sub-population. CONCLUSIONS: Switching to maintenance using ZKAB001 showed an acceptable safety profile and provided preliminary evidence of clinical activity in localized patients with osteosarcoma.

Analysis of human Ki-67 gene promoter and identification of the Sp1 binding sites for Ki-67 transcription.

Ki-67 as a cell proliferation marker is tightly associated with maintenance and regulation of the cell division. To understand the mechanism of Ki-67 gene expression and regulation, we first cloned 5'-flanking region and identified the Ki-67 core promoter. The deletion analysis and the dual luciferase reporter assay were used to locate the Ki-67 core promoter from -223 to +12 nt relative to the transcriptional initiation site, which is the TATA less, GC rich region comprised of several putative Sp1 binding sites. Compared with the hTERT promoter and Survivin promoter, the Ki-67 core promoter possessed higher transcription activity and more desirable tumor selectivity. In order to further demonstrate the contribution of transcription factor Sp1 on regulating the Ki-67 gene transcription, we confirmed three Sp1 binding sites from -170 to -145 nt, from -63 to -38 nt, and from -14 to +12 nt existed in the Ki-67 core promoter by the supershift assay. The deletion mutagenesis, together with the dual luciferase reporter assay, indicated that these Sp1 binding sites, particularly the region from -170 to -145 nt, were involved in positive regulation of the Ki-67 gene expression. Collectively, it was demonstrated that the region from -223 to +12 nt could drive the transcription of the Ki-67 gene, and the Sp1 binding site is essential to transcriptional regulation of the Ki-67 gene.

Surgical Management of Cardiac Masses in Right Atrium Among Bone Sarcoma Pediatric Patients With Totally Implanted Ports.

Introduction: Totally implanted ports (PORTs) have been widely used among patients with malignancy. Cardiac metastasis secondary to bone sarcoma and catheter-related right atrial thrombosis (CRAT) can be both present as cardiac masses. However, these two cardiac masses share very similar imaging characteristics. Methods: The features, treatments, and outcomes of 5 bone sarcoma pediatric patients with PORTs who suffered from cardiac masses in the right atrium were analyzed. Clinical data and histological characteristics of cardiac masses were also recorded. Results: Among 928 patients with malignancy and PORTs, 5 bone sarcoma pediatric patients were found to have cardiac masses in the right atrium. The catheter tips were located in the right atrium of 4 patients and the superior vena cava-right atrium junction (CAJ) of 1 patient. Four patients with good response to anti-tumor treatment had received surgical lumpectomies for pathologic identification and mass excision, with cardiac metastases among 1 patient and thromboses among 3 patients. The median time from venous access port implantation to cardiac mass detection for CRAT was 6.3 months (range: 4.7-6.8 months) and to diagnosis of or possible cardiac metastasis was 13.3 months (range: 11.2-15.4 months). Conclusion: The placement of a catheter tip into the right atrium should be avoided. The time from PORTs implantation to cardiac mass detection might serve as a potential tool to differentiate cardiac metastasis from CRAT. Surgical management may be an effective treatment for bone sarcoma pediatric patients who had good response to anti-tumor treatment and suffered from cardiac masses in the right atrium.

The Combination of Anlotinib and Gemcitabine/Docetaxel in Patients with Metastatic Osteosarcoma Who Have Failed Standard Chemotherapy.

Purpose: The options for the second-line treatment of metastatic osteosarcoma are still limited. Anlotinib is a multi-kinase inhibitor which has shown promising efficacy and good tolerability in various cancer types. This retrospective study was conducted to evaluate the efficacy and safety of anlotinib combined with gemcitabine/docetaxel (GD) in patients with metastatic osteosarcoma who have failed first-line chemotherapy. Patients and Methods: The data of patients who received anlotinib combined with GD or GD were collected. The primary endpoint was progression-free survival. Secondary endpoints included objective response rate and safety. Results: From July 2013 to November 2020, a total of 32 patients were enrolled, 13 received anlotinib combined with GD and 19 received GD. Median PFS was 9.0 months (95% CI 6.7-39.1) in the combination group and 5.0 months (95% CI 1.2-6.7) in the chemotherapy group. ORR were 38.4% and 15.8%, DCR were 69.2% and 38.1% in the combination and chemotherapy group, respectively. The most common adverse events included fatigue (78.9% in the combination group vs 69.2% in the chemotherapy group), hypertension (46.2% vs 10.5%), diarrhea (38.5% vs 21.1%), hypothyroidism (38.5% vs 15.8%), neutropenia (23.1% vs 36.8%) and AST elevation (30.8% vs 21.1%). The most common grade 3 or worse adverse events included hand-foot reaction (7.7% vs 5.3%), hypothyroidism (15.4% vs 0), neutropenia (0 vs 10.5%). Conclusion: The combination of anlotinib and GD showed favorable efficacy with manageable toxicities compared with GD in the second-line treatment for metastatic osteosarcoma. This combination therapy deserves further investigations in patients with osteosarcoma.

Fibrinogen-Albumin Ratio Index Exhibits Predictive Value of Neoadjuvant Chemotherapy in Osteosarcoma.

Purpose: Inflammatory response and nutritional status are associated with cancer development and progression. The present study aimed to evaluate the predictive ability of the fibrinogen-albumin ratio index (FARI) to the efficacy of neoadjuvant chemotherapy (NAC) for osteosarcoma. Patients and methods: A retrospective analysis involving 752 consecutive osteosarcoma patients between 2012 and 2020 was performed. Data on serum fibrinogen, albumin levels, white blood cell count, platelet count, and alkaline phosphatase (ALP) before and after NAC were collected. The predictive value of the NAC efficacy in osteosarcoma was assessed by constructing a receiver operating characteristic (ROC) curve and calculating the area under the curve (AUC). Prognosis and its predictive factors were analyzed by Kaplan-Meier method and COX regression analysis. Nomogram was established according to selected variables. The predictive performance of the nomogram model was assessed using C-statistics. Results: A total of 203 patients were included. ROC analysis showed that both FARI before NAC (preFARI; AUC = 0.594, p = 0.032) and the change in FARI before and after NAC (dfFARI = preFARI-postFARI; AUC = 0.652, p = 0.001) exhibited more favorable predictive ability than ALP and other inflammation markers. The preFARI was divided into the high group (>6.1%) and the low group (≤6.1%) based on the optimal cut-off value of 6.1%. Patients with a high preFARI showed significantly decreased metastasis-free survival (MFS) and disease-free survival (DFS) (all p<0.01). In multivariable analysis, preFARI was an independent prognostic marker for patients with osteosarcoma. Predictive nomograms exhibited good ability to predict MFS (C-index = 0.748, se = 0.028) and DFS (C-index=0.727, se = 0.030). Conclusion: Our findings indicated that FARI exhibits the favorable predictive ability for the efficacy of NAC for osteosarcoma, which could support clinicians and patients in clinical decision-making and treatment optimization.

A critical role of Sp1 transcription factor in regulating the human Ki-67 gene expression.

Ki-67 plays a crucial role in cell proliferation as well as maintenance or regulation of cell division. The mechanism governing the Ki-67 gene expression remains unknown. Thus, we cloned the core promoter of the human Ki-67 gene and further investigated its transcriptional regulation. The putative Sp1 binding sites were confirmed by electrophoretic mobility shift assay together with an anti-Sp1 antibody-mediated supershift assay. Deletion mutagenesis and firefly luciferase reporter gene assay demonstrated the essential contribution of Sp1 on transcriptional activation of the Ki-67 gene. In this study, we first confirm that there are three Sp1 binding sites in the Ki-67 core promoter. Two Sp1 sites (one at position -159 to -145 nt and the other at position -14 to +12 nt) are mainly involved in transcriptional regulation of the Ki-67 gene. Overexpression of Sp1 can enhance the Ki-67 promoter activity. However, down-regulation of Sp1 expression using siRNA-Sp1 and mithramycin effectively inhibits the Ki-67 gene transcription. Our results suggest that Sp1 is essential for basal promoter activity of the human Ki-67 gene. Inhibition of the Ki-67 transcriptional activity through abolishment of Sp1 may provide the useful prospect for gene therapy.

p53 regulates Ki-67 promoter activity through p53- and Sp1-dependent manner in HeLa cells.

The expression of the human Ki-67 protein, which is strictly associated with cell proliferation, is regulated by a variety of cellular mediators. In this study, we studied the effects of p53 on Ki-67 promoter in HeLa cells using luciferase reporter assay. The results showed that: (1) p53 inhibited Ki-67 promoter activity in a dose-dependent manner, (2) the p53-binding motifs mediated part of the transcriptional repression of Ki-67 promoter through a sequence-specific interaction with p53, (3) p53 was able to repress the Sp1-stimulated Ki-67 promoter activity, and (4) the Sp1-binding sites were responsible for the p53-mediated transcriptional repression of Ki-67 promoter. In conclusion, p53 inhibited Ki-67 promoter activity via p53- and Sp1-dependent pathways, and the interaction between p53 and Sp1 might be involved in the transcriptional regulatory mechanisms.

The effect of methylated oligonucleotide targeting Ki-67 gene in human 786-0 renal carcinoma cells.

To investigate the effect of methylated oligonucleotide (MON) targeting Ki-67 promoter on the expression of Ki-67 gene and the proliferation and apoptosis of the human 786-0 renal carcinoma cells, human 786-0 cells were transfected with MON. The activity of Ki-67 promoter was detected by dual-luciferase reporter assay system. Among the five methylated oligonucleotides (MON(1)-MON(5)), MON(4) is the best excellent one in the inhibition of the Ki-67 promoter activity. The activity of Ki-67 promoter is decreased to 77.88% in 40-nM group, 50.07% in 80-nM group, 35.63% in 120-nM group, 26.09% in 160-nM group, and 16.98% in 200-nM group compared with 0-nM group. The activity of Ki-67 promoter in MON group is decreased to 61.96% at 8 h, 48.93% at 12 h, 15.97% at 24 h, 26.00% at 36 h, 35.01% at 48 h, 46.08% at 72 h, and 66.12% at 96 h compared with pGLBK235 group. These results show that the effect of MON is time- and dose-dependent. The activity of Ki-67 in MON group is decreased to 16.73% compared with pGLBK235 group, while the control groups have no significant difference. The expression of Ki-67 gene in 786-0 cells was detected by RT-PCR and immunohistochemistry, respectively. The expression of Ki-67 mRNA is decreased to 61.04% and that of Ki-67 protein is decreased to 32.07% in MON group compared with the blank group. The proliferation of 786-0 cells was determined by WST-8. The cell proliferation in MON group is decreased to 61.02% at 24 h, 73.78% at 48 h, 79.72% at 72 h, and 91.53% at 96 h compared with the blank group. The cell apoptosis was measured by annexin V and propidium iodide. The number of apoptosis cells in MON group is 2.42 times of that in the blank group at earlier period and 2.57 times at mid-anaphase. We detected the effect of MON on the expression of bax and p53 by Western blot. Compared with the blank group, the expression of bax protein in MON group is increased by 66.12%, while the expression of p53 is decreased to 67.31%. Our study demonstrates that the methylated oligonucleotide targeting Ki-67 promoter has a remarkable effect on the inhibition of Ki-67 expression and the proliferation of the human 786-0 renal carcinoma cells and can induce apoptosis of the 786-0 cells.

Case Report: Sequential Chemotherapy and Immunotherapy Produce Sustained Response in Osteosarcoma With High Tumor Mutational Burden.

Background: Immunotherapy has provided an effective method for the treatment of many cancers. However, its efficacy in osteosarcoma is not satisfactory so far. Case Presentation: Here, we presented a case of osteosarcoma treated with sequential chemotherapy and immunotherapy and showed promising therapeutic potential. The 29-year-old female patient presented 9th rib osteosarcoma with suspected right lung lower lobe metastasis. Surgery was performed to remove the primary lesion, and a series of chemotherapies were given afterward in consideration of the response and tolerance. The right lung lower lobe metastasis was under control first but progressed (PD) 9 months after the initiation of therapy. The lesion was surgically removed and subsequent chemotherapy was implemented. The patient had good tolerance with chemotherapy and maintained well for approximately 11 months before the discovery of 11th rib and right lung upper lobe metastases. Surgery was then performed on both lesions and achieved complete response. Post-surgical brief chemotherapy and subsequent long-term immunotherapy (pembrolizumab) maintained continuous remission for 33 months. The patient survived for 60 months with well-controlled disease from the time of confirmed diagnosis. Genetic alterations of all primary and metastatic lesions were investigated by whole-exome sequencing (WES). Substantial similarity in mutational landscape between the primary lesion and 11th rib metastasis and between the two lung metastases were revealed, while substantial heterogeneity was found between the rib lesions and lung metastases. The tumor mutational burden (TMB) for the 9th rib primary lesion, the metastatic 11th rib lesion, and the metastatic right upper and lower lobe nodule tissues was 8.02, 2.38, 4.61, and 0.14 mutations/Mb, respectively. The primary lesion exhibited the most diverse copy number variation (CNV) changes among all lesions. Furthermore, pathway enrichment analysis also suggested significant heterogeneity among the lesions. Conclusions: Surgery with sequential chemotherapy and maintenance immunotherapy was shown to have good response for the first time on osteosarcoma patient who had high TMB tumor lesions and good tolerance for chemotherapy and immunotherapy.

N6-Methyladenosine modification of the TRIM7 positively regulates tumorigenesis and chemoresistance in osteosarcoma through ubiquitination of BRMS1.

BACKGROUND: Metastasis is the leading cause of death in patients with osteosarcoma. Some of these patients fail to respond to chemotherapy and die of metastasis within a short period. Therefore, it is important to identify novel biomarkers to improve the diagnosis and treatment of osteosarcoma. TRIM7 is a member of the tripartite motif (TRIM) family protein that is involved in various pathological conditions including cancer; however, its role in osteosarcoma remains elusive. METHODS: Cell proliferation, invasion and migration were measured by CCK-8 and Transwell. Immunoprecipitation and mass spectrometry analysis were used to identify candidate proteins associated with TRIM7. Immunoprecipitation, immunofluorescence, pull down and ubiquitination assay were performed to examine the regulation between TRIM7 and its candidate protein. m6A modification of TRIM7 was measured by RNA immunoprecipitation. FINDINGS: TRIM7 expression was upregulated in osteosarcoma tissues and was an independent risk factor in predicting poor prognosis. TRIM7 regulates osteosarcoma cell migration and invasion through ubiquitination of breast cancer metastasis suppressor 1 (BRMS1). Moreover, chemoresistance was readily observed in osteosarcoma cells and in patient-derived xenograft (PDX) mice with higher TRIM7 levels. Loss of TRIM7 m6A modification was observed in osteosarcoma tissues. METTL3 and YTHDF2 were the main factors involved in the aberrant m6A modification of TRIM7. INTERPRETATION: Overall, our findings show that TRIM7 plays a key role in regulating metastasis and chemoresistance in osteosarcoma through ubiquitination of BRMS1. FUNDING: This work was financially supported by grants of NSFC (81001192, 81672658 and 81972521) and National Key Research Project of Science and Technology Ministry (2016YFC0106204).

Visfatin is involved in the cisplatin resistance of osteosarcoma cells via upregulation of Snail and Zeb1.

Understanding the mechanisms of chemoresistance in osteosarcoma (OS) cell is important for drug development. By establishment of cisplatin (CDDP) resistant OS cells, we found that the levels of visfatin in OS/CDDP cells were significantly greater than that in their parental cells. The CDDP resistant OS cells showed greater migration and invasion capability than that of parental cells. Knockdown of visfatin can rescue the CDDP sensitivity of resistant OS cells. Among the detected epithelial-mesenchymal transition-related transcription factors (EMT-TFs), visfatin can increase the expression of Snail and Zeb-1 in OS cells. Overexpression of Snail and Zeb1 can attenuate si-visfatin reduced CDDP resistance of OS cells. Mechanistical studies indicated that visfatin can increase the mRNA expression of Snail and therefore upregulate its expression via HIF-1α induced transcription. As to Zeb1, visfatin had no effect on its mRNA expression, while significantly increased its protein stability. Furthermore, the upregulation of ATM, which can phosphorylate and stabilize Zeb1, was involved in visfatin-induced Zeb1 expression in OS cells. Collectively, our revealed that visfatin was involved in CDDP resistance of OS cells via upregulation of Snail and Zeb1, suggesting that inhibition of visfatin might be a potential pathway for OS treatment.

Reciprocal Role Of DNA Methylation And Sp1 Binding In Ki-67 Gene Transcription.

PURPOSE: DNA methylation plays major regulatory roles in gene transcription. Our previous studies confirmed that Ki-67 promoter is hypomethylated and Sp1 is a transcriptional activator of Ki-67 gene in cancer cells. However, whether Sp1-mediated transcriptional activation of Ki-67 is related to its methylation has not been studied yet. MATERIALS AND METHODS: In this study, we confirmed that methylated CpG binding protein 2 (MBD2) binding to methylated DNA hindered the binding of Sp1 to Ki-67 promoter and then repressed Ki-67 transcription through chromatin immunoprecipitation (ChIP) and quantitative real-time PCR (qRT-PCR). Co-immunoprecipitation (Co-IP), ChIP, methylation-specific PCR (MS-PCR) and Western blot were utilized to analyze the effects of Sp1 binding to Ki-67 promoter on its methylation status. RESULTS: Less DNA methyltransferase 1 (DNMT1) bound to the Ki-67 promoter in MKN45 cells than in HK-2 cells. Histone acetyltransferase p300 that was recruited by Sp1 to Ki-67 promoter could attenuate the methylation level of Ki-67 promoter. Furthermore, higher expression of Sp1 and Ki-67 was related to the overall survival (OS), first progression (FP) and post-progression survival (PPS) in gastric cancer by scrutinizing bioinformatics datasets. CONCLUSION: Taken together, our findings suggested that hypomethylation of Ki-67 promoter enhanced the binding of Sp1, which in turn maintained hypomethylation of promoter, leading to increase Ki-67 expression in cancer cells. Sp1 and Ki-67 could act promising prognostic biomarkers for clinical diagnosis and treatment of cancer.

Effect of radiotherapy on survival of women with locally excised ductal carcinoma in situ of the breast: a Surveillance, Epidemiology, and End Results population-based analysis.

BACKGROUND: Although it has been previously reported that radiotherapy (RT) effectively reduced the incidence of local recurrence of ductal carcinoma in situ (DCIS) following breast-conserving surgery (BCS), little is known about the effect of RT on survival of patients with locally excised DCIS. PATIENTS AND METHODS: Using Surveillance, Epidemiology, and End Results registry data, we selected 56,968 female DCIS patients treated with BCS between 1998 and 2007. Overall survival (OS) and breast cancer-specific survival (BCSS) were compared among patients who received RT or no RT using the Kaplan-Meier methods and Cox proportional hazards regression models. RESULTS: Median follow-up was 91 months. In the multivariable model, patients receiving postoperative RT had better OS than those undergoing BCS alone (hazard ratio [HR] 0.59, 95% confidence interval [CI] 0.53-0.67, P<0.001). This pattern remained after stratification by estrogen receptor (ER) status and age. In contrast, RT delivery was not significantly associated with improved BCSS (HR 0.71, 95% CI 0.48-1.03, P=0.073). However, after stratifying by the above two variables, RT contributed to better BCSS in ER-negative/borderline patients (HR 0.41, 95% CI 0.19-0.88, P=0.023) and younger patients (≤50 years old; HR 0.37, 95% CI 0.15-0.91, P=0.030). CONCLUSION: Our analysis confirms the beneficial effect of RT on OS in women with locally excised DCIS and reveals the specific protective effect of RT on BCSS in ER-negative/borderline and younger patients.

An elevated peripheral blood lymphocyte-to-monocyte ratio predicts favorable response and prognosis in locally advanced breast cancer following neoadjuvant chemotherapy.

PURPOSE: Neoadjuvant chemotherapy (NCT) is a standard treatment option for locally advanced breast cancer. However, the lack of an efficient method to predict treatment response and patient prognosis hampers the clinical evaluation of patient eligibility for NCT. An elevated lymphocyte-to-monocyte ratio (LMR) has been reported to be associated with a favorable prognosis for certain hematologic malignancies and for nasopharyngeal carcinoma; however, this association has not been investigated in breast cancer. The purpose of this study was to evaluate whether pre-NCT LMR analysis could predict the prognosis of patients with locally advanced breast cancer. METHODS: A retrospective cohort of 542 locally advanced breast cancer patients (T3/T4 and/or N2/N3 disease) receiving NCT followed by radical surgery was recruited between May 2002 and August 2011 at the Fudan University Shanghai Cancer Center. Counts for pre-NCT peripheral absolute lymphocytes and monocytes were obtained and used to calculate the LMR. RESULTS: Univariate and multivariate analysis revealed that higher LMR levels (≥4.25) were significantly associated with favorable DFS (P = 0.009 and P = 0.011, respectively). Additionally, univariate analysis revealed that a higher lymphocyte count (≥1.5×109/L) showed borderline significance for improved DFS (P = 0.054), while a lower monocyte count (<0.4×109/L) was associated with a significantly better DFS (P = 0.010). CONCLUSIONS: An elevated pre-NCT peripheral LMR level was a significantly favorable factor for locally advanced breast cancer patient prognosis. This easily obtained variable may serve as a valuable marker to predict the outcomes of locally advanced breast cancer.