A novel long non-coding RNA MIR4500HG003 promotes tumor metastasis through miR-483-3p-MMP9 axis in triple-negative breast cancer.

Breast cancer (BC) is the most common cancer and the leading cause of cancer-related deaths in women worldwide. The 5-year survival rate is over 90% in BC patients, but once BC cells metastasis into distal organs, it is dramatically decreasing to less than 30%. Especially, triple-negative breast cancer (TNBC) patients usually lead to poor prognosis and survival because of metastasis. Understanding the underline mechanisms of TNBC metastasis is a critical issue. Non-coding RNAs, including of lncRNAs and microRNAs, are non-protein-coding transcripts and have been reported as important regulators in TNBC metastasis. However, the underline mechanisms for non-coding RNAs regulating TNBC metastasis remain largely unclear. Here, we found that lncRNA MIR4500HG003 was highly expressed in highly metastatic MDA-MB-231 TNBC cells and overexpression of MIR4500HG003 enhanced metastasis ability in vitro and in vivo and promoted MMP9 expression. Furthermore, we found MIR4500HG003 physically interacted with miR-483-3p and reporter assay showed miR-483-3p attenuated MMP9 expression. Importantly, endogenous high expressions of MIR4500HG003 were correlated with tumor recurrence in TNBC patients with tumor metastasis. Taken together, our findings suggested that MIR4500HG003 promotes metastasis of TNBC through miR-483-3p-MMP9 signaling axis and may be used as potential prognostic marker for TNBC patients.

ICAM2 initiates trans-blood-CSF barrier migration and stemness properties in leptomeningeal metastasis of triple-negative breast cancer.

Leptomeningeal metastasis (LM) occurs when tumor cells spread to the leptomeningeal space surrounding the brain and the spinal cord, thereby causing poor clinical outcomes. The triple-negative breast cancer (TNBC) has been associated with symptoms of LM and mechanism remained unclear. Through proteomic analysis, we identified high expression of ICAM2 in leptomeningeal metastatic TNBC cells, which promoted the colonization of the spinal cord and resulted in poor survival in vivo. Two-way demonstration indicated that high levels of ICAM2 promoted blood-cerebrospinal fluid barrier (BCB) adhesion, trans-BCB migration, and stemness abilities and determined the specificity of LM in vivo. Furthermore, pull-down and antibody neutralizing assay revealed that ICAM2 determined the specificity of LM through interactions with ICAM1 in the choroid plexus epithelial cells. Therefore, neutralizing ICAM2 can attenuate the progression of LM and prolong survival in vivo. The results suggested that targeting ICAM2 is a potential therapeutic strategy for LM in TNBC.

Carboxyl-terminal modulator protein facilitates tumor metastasis in triple-negative breast cancer.

Currently, the survival rate for breast cancer is more than 90%, but once the cancer cells metastasize to distal organs, the survival rate is dramatically reduced, to less than 30%. Triple-negative breast cancer accounts for 15-20% of all breast cancers. Triple-negative breast cancer (TNBC) is associated with poor prognostic and diagnostic outcomes due to the limiting therapeutic strategies, relative to non-TNBC breast cancers. Therefore, the development of targeted therapy for TNBC metastasis remains an urgent issue. In this study, high Carboxyl-terminal modulator protein (CTMP) is significantly associated with recurrence and disease-free survival rate in TNBC patients. Overexpression of CTMP promotes migration and invasion abilities in BT549 cells. Down-regulating of CTMP expression inhibits migration and invasion abilities in MDA-MB-231 cells. In vivo inoculation of high-CTMP cells enhances distant metastasis in mice. The metastasis incidence rate is decreased in mice injected with CTMP-downregulating MDA-MB-231 cells. Gene expression microarray analysis indicates the Akt-dependent pathway is significantly enhanced in CTMP overexpressing cells compared to the parental cells. Blocking Akt activation via Akt inhibitor treatment or co-expression of the dominant-negative form of Akt proteins successfully abolishes the CTMP mediating invasion in TNBC cells. Our findings suggest that CTMP is a potential diagnostic marker for recurrence and poor disease-free survival in TNBC patients. CTMP promotes TNBC metastasis via the Akt-activation-dependent pathway.

Concurrent Chemoradiotherapy-Driven Cell Plasticity by miR-200 Family Implicates the Therapeutic Response of Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a common and fatal malignancy with an increasing incidence worldwide. Over the past decade, concurrent chemoradiotherapy (CCRT) with or without surgery is an emerging therapeutic approach for locally advanced ESCC. Unfortunately, many patients exhibit poor response or develop acquired resistance to CCRT. Once resistance occurs, the overall survival rate drops down rapidly and without proper further treatment options, poses a critical clinical challenge for ESCC therapy. Here, we utilized lab-created CCRT-resistant cells as a preclinical study model to investigate the association of chemoradioresistantresistance with miRNA-mediated cell plasticity alteration, and to determine whether reversing EMT status can re-sensitize refractory cancer cells to CCRT response. During the CCRT treatment course, refractory cancer cells adopted the conversion of epithelial to mesenchymal phenotype; additionally, miR-200 family members were found significantly down-regulated in CCRT resistance cells by miRNA microarray screening. Down-regulated miR-200 family in CCRT resistance cells suppressed E-cadherin expression through snail and slug, and accompany with an increase in N-cadherin. Rescuing expressions of miR-200 family members in CCRT resistance cells, particularly in miR-200b and miR-200c, could convert cells to epithelial phenotype by increasing E-cadherin expression and sensitize cells to CCRT treatment. Conversely, the suppression of miR-200b and miR-200c in ESCC cells attenuated E-cadherin, and that converted cells to mesenchymal type by elevating N-cadherin expression, and impaired cell sensitivity to CCRT treatment. Moreover, the results of ESCC specimens staining established the clinical relevance that higher N-cadherin expression levels associate with the poor CCRT response outcome in ESCC patients. Conclusively, miR-200b and miR-200c can modulate the conversion of epithelial-mesenchymal phenotype in ESCC, and thereby altering the response of cells to CCRT treatment. Targeting epithelial-mesenchymal conversion in acquired CCRT resistance may be a potential therapeutic option for ESCC patients.

Synergy between the pay-for-performance scheme and better physician-patient relationship might reduce the risk of retinopathy in patients with type 2 diabetes.

AIMS/INTRODUCTION: This study investigated whether participation by patients with type 2 diabetes in Taiwan's pay-for-performance (P4P) program and maintaining good continuity of care (COC) with their healthcare provider reduced the likelihood of future complications, such as retinopathy. MATERIALS AND METHODS: The analysis used longitudinal panel data for newly diagnosed type 2 diabetes from the National Health Insurance claims database in Taiwan. COC was measured annually from 2003 to 2013, and was used to allocate the patients to low, medium and high groups. Cox regression analysis was used with time-dependent (time-varying) covariates in a reduced model (with only P4P or COC), and the full model was adjusted with other covariates. RESULTS: Despite the same significant effects of treatment at primary care, the Diabetes Complications Severity Index scores were significantly associated with the development of retinopathy. After adjusting for these, the hazard ratios for developing retinopathy among P4P participants in the low, medium and high COC groups were 0.594 (95% confidence interval [CI] 0.398-0.898, P = 0.012), 0.676 (95% CI 0.520-0.867, P = 0.0026) and 0.802 (95% CI 0.603-1.030, P = 0.1062), respectively. Thus, patients with low or median COC who participated in the P4P program had a significantly lower risk of retinopathy than those who did not. CONCLUSIONS: Diabetes care requires a long-term relationship between patients and their care providers. Besides encouraging patients to participate in P4P programs, health authorities should provide more incentives for providers or patients to regularly survey patients' lipid profiles and glucose levels, and reward the better interpersonal relationship to prevent retinopathy.

Sialylation of CD55 by ST3GAL1 Facilitates Immune Evasion in Cancer.

Altered glycosylations, which are associated with expression and activities of glycosyltransferases, can dramatically affect the function of glycoproteins and modify the behavior of tumor cells. ST3GAL1 is a sialyltransferase that adds sialic acid to core 1 glycans, thereby terminating glycan chain extension. In breast carcinomas, overexpression of ST3GAL1 promotes tumorigenesis and correlates with increased tumor grade. In pursuing the role of ST3GAL1 in breast cancer using ST3GAL1-siRNA to knockdown ST3GAL1, we identified CD55 to be one of the potential target proteins of ST3GAL1. CD55 is an important complement regulatory protein, preventing cells from complement-mediated cytotoxicity. CD55 had one N-linked glycosylation site in addition to a Ser/Thr-rich domain, which was expected to be heavily O-glycosylated. Detailed analyses of N- and O-linked oligosaccharides of CD55 released from scramble or ST3GAL1 siRNA-treated breast cancer cells by tandem mass spectrometry revealed that the N-glycan profile was not affected by ST3GAL1 silencing. The O-glycan profile of CD55 demonstrated a shift in abundance to nonsialylated core 1 and monosialylated core 2 at the expense of the disialylated core 2 structure after ST3GAL1 silencing. We also demonstrated that O-linked desialylation of CD55 by ST3GAL1 silencing resulted in increased C3 deposition and complement-mediated lysis of breast cancer cells and enhanced sensitivity to antibody-dependent cell-mediated cytotoxicity. These data demonstrated that ST3GAL1-mediated O-linked sialylation of CD55 acts like an immune checkpoint molecule for cancer cells to evade immune attack and that inhibition of ST3GAL1 is a potential strategy to block CD55-mediated immune evasion.

Sialylation of vasorin by ST3Gal1 facilitates TGF-ß1-mediated tumor angiogenesis and progression.

ST3Gal1 is a key sialyltransferase which adds α2,3-linked sialic acid to substrates and generates core 1 O-glycan structure. Upregulation of ST3Gal1 has been associated with worse prognosis of breast cancer patients. However, the protein substrates of ST3Gal1 implicated in tumor progression remain elusive. In our study, we demonstrated that ST3GAL1-silencing significantly reduced tumor growth along with a notable decrease in vascularity of MCF7 xenograft tumors. We identified vasorin (VASN) which was shown to bind TGF-ß1, as a potential candidate that links ST3Gal1 to angiogenesis. LC-MS/MS analysis of VASN secreted from MCF7, revealed that more than 80% of its O-glycans are sialyl-3T and disialyl-T. ST3GAL1-silencing or desialylation of VASN by neuraminidase enhanced its binding to TGF-ß1 by 2- to 3-fold and thereby dampening TGF-ß1 signaling and angiogenesis, as indicated by impaired tube formation of HUVECs, suppressed angiogenesis gene expression and reduced activation of Smad2 and Smad3 in HUVEC cells. Examination of 114 fresh primary breast cancer and their adjacent normal tissues showed that the expression levels of ST3Gal1 and TGFB1 were high in tumor part and the expression of two genes was positively correlated. Kaplan Meier survival analysis showed a significantly shorter relapse-free survival for those with lower expression VASN, notably, the combination of low VASN with high ST3GAL1 yielded even higher risk of recurrence (p = 0.025, HR = 2.967, 95% CI = 1.14-7.67). Since TGF-ß1 is known to transcriptionally activate ST3Gal1, our findings illustrated a feedback regulatory loop in which TGF-ß1 upregulates ST3Gal1 to circumvent the negative impact of VASN.

Reciprocal feedback regulation of ST3GAL1 and GFRA1 signaling in breast cancer cells.

GFRA1 and RET are overexpressed in estrogen receptor (ER)-positive breast cancers. Binding of GDNF to GFRA1 triggers RET signaling leading to ER phosphorylation and estrogen-independent transcriptional activation of ER-dependent genes. Both GFRA1 and RET are membrane proteins which are N-glycosylated but no O-linked sialylation site on GFRA1 or RET has been reported. We found GFRA1 to be a substrate of ST3GAL1-mediated O-linked sialylation, which is crucial to GDNF-induced signaling in ER-positive breast cancer cells. Silencing ST3GAL1 in breast cancer cells reduced GDNF-induced phosphorylation of RET, AKT and ERα, as well as GDNF-mediated cell proliferation. Moreover, GDNF induced transcription of ST3GAL1, revealing a positive feedback loop regulating ST3GAL1 and GDNF/GFRA1/RET signaling in breast cancers. Finally, we demonstrated ST3GAL1 knockdown augments anti-cancer efficacy of inhibitors of RET and/or ER. Moreover, high expression of ST3GAL1 was associated with poor clinical outcome in patients with late stage breast cancer and high expression of both ST3GAL1 and GFRA1 adversely impacted outcome in those with high grade tumors.

Assessment of duration until initial treatment and its determining factors among newly diagnosed oral cancer patients: A population-based retrospective cohort study.

Few studies have focused on the early treatment stages of cancer, and the impact of treatment delay on oncologic outcomes is poorly defined. We used oral cancer as an example to investigate the distribution of durations until initial treatment.This study was conducted using the National Health Insurance Research Database, which is linked to Taiwan's Cancer Registry and Death Registry databases. We defined "cutoff points for first-time treatment" according to a weekly schedule and sorted the patients into 2 groups based on whether their duration until initial treatment was longer or shorter than each cutoff. We then calculated the Kaplan-Meier estimator to determine the difference in survival rates between the 2 groups and performed logistic regression to identify determining factors.The average time between diagnosis and initial treatment was approximately 22.45 days. The average survival duration was 1363 days (standard deviation: 473.06 days). Oral cancer patients had no statistically significant differences in survival until a cutoff point of 3 weeks was used (with survival duration 71 days longer if initial treatment was received within 3 weeks). Patients with higher incomes or higher Charlson comorbidity index scores and patients treated at a hospital in a region with medium urbanization had lower likelihoods of treatment delay, whereas older patients were at higher risk of treatment delay.The attitudes, beliefs, and social contexts of oral cancer patients influence the treatment-seeking behaviors of these patients. Therefore, the government should advocate the merits of the referral system for cancer treatment or improve quality assurance for cancer diagnoses across different types of hospitals. Health authorities should also educate patients or use a case manager to encourage prompt treatment within 3 weeks and should provide screening and prevention services, particularly for high-risk groups, to reduce mortality risk.

The relationship between accessibility of healthcare facilities and medical care utilization among the middle-aged and elderly population in Taiwan.

OBJECTIVE: The purpose of this study was to explore the relationship between accessibility of healthcare facilities and medical care utilization among the middle-aged and elderly population in Taiwan. DESIGN: Cross-sectional study from 2007 Taiwan Longitudinal Study on Ageing (TLSA) survey. SETTING: Community-based study. PARTICIPANTS: A total of 4249 middle-aged and elderly subjects were recruited. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Outpatient visits within 1 month, and hospitalization, emergency visits as well as to shop in pharmacy stores within 1 year, respectively. RESULTS: Adjusting for important confounding variables, the middle-aged and elderly with National Health Insurance (NHI) and commercial insurance compared with those with NHI alone tended to have outpatient visits. The middle-aged and elderly with longer time to access healthcare facilities were less likely to shop in pharmacy stores compared with those with <30 min. The middle-aged and elderly who perceived inconvenient to access health care tended to shop in pharmacy stores compared with those with perceived convenience. CONCLUSIONS: Our study of Taiwan's experience could provide a valuable lesson for countries that are planning to launch universal health insurance system, locate budgets in health care and transportation. The middle-aged and elderly who were facing more challenges in accessing health care, no matter in perceived accessibility or real time to access health care, had less outpatient visits and more drug stores shopping. Strategic policies are needed to improve accessibility in increasing patients' perception on access and escalating convenience of transportation system for improving accessibility.

Chimeras of p14ARF and p16: functional hybrids with the ability to arrest growth.

The INK4A locus codes for two independent tumor suppressors, p14ARF and p16/CDKN2A, and is frequently mutated in many cancers. Here we report a novel deletion/substitution from CC to T in the shared exon 2 of p14ARF/p16 in a melanoma cell line. This mutation aligns the reading frames of p14ARF and p16 mid-transcript, producing one protein which is half p14ARF and half p16, chimera ARF (chARF), and another which is half p16 and half non-p14ARF/non-p16 amino acids, p16-Alternate Carboxyl Terminal (p16-ACT). In an effort to understand the cellular impact of this novel mutation and others like it, we expressed the two protein products in a tumor cell line and analyzed common p14ARF and p16 pathways, including the p53/p21 and CDK4/cyclin D1 pathways, as well as the influence of the two proteins on growth and the cell cycle. We report that chARF mimicked wild-type p14ARF by inducing the p53/p21 pathway, inhibiting cell growth through G2/M arrest and maintaining a certain percentage of cells in G1 during nocodazole-induced G2 arrest. chARF also demonstrated p16 activity by binding CDK4. However, rather than preventing cyclin D1 from binding CDK4, chARF stabilized this interaction through p21 which bound CDK4. p16-ACT had no p16-related function as it was unable to inhibit cyclin D1/CDK4 complex formation and was unable to arrest the cell cycle, though it did inhibit colony formation. We conclude that these novel chimeric proteins, which are very similar to predicted p16/p14ARF chimeric proteins found in other primary cancers, result in maintained p14ARF-p53-p21 signaling while p16-dependent CDK4 inhibition is lost.

Continuity of diabetes care is associated with avoidable hospitalizations: evidence from Taiwan's National Health Insurance scheme.

OBJECTIVE: Taiwan's health-care system allows patients to utilize specialty services without referrals by primary care providers. This discontinuity of care may lead to increases in future hospitalizations. This study aims to determine whether the discontinuity of care is associated with the risk of hospitalization. DESIGN: A secondary data analysis based on a claim data of a nationally representative random sample of diabetic patients in Taiwan. A usual provider continuity (UPC) index was developed-a ratio of the visits to the physician that subjects most usually see relevant to diabetes care to the total physician visits relevant to diabetes care-to investigate its association with the risk of hospitalization. SETTING: Taiwan's National Health Insurance scheme from 1997 through 2002. PARTICIPANTS: Totally 6476 diabetic patients. INTERVENTION(s) None. MAIN OUTCOME MEASURE(s) Diabetes-related short-term and long-term ambulatory care sensitive condition (ACSC) admissions. RESULTS: Patients with ACSC admissions had significantly lower UPC scores compared with those without ACSC admissions. Using a Cox regression model that controlling for age, sex, severity of diabetes and the number of total visits, patients with low to medium continuity of care (UPC <0.75) were found to be significantly associated with increased risk of hospitalization as compared with patients with high continuity of care, especially for long-term ACSC admissions (relative risk: 1.336 [1.019-1.751]). CONCLUSIONS: Higher continuity of care with usual providers for diabetic care is significantly associated with lower risk of future hospitalization for long-term diabetic complication admissions. To avoid future hospitalization, health policy stakeholders are encouraged to improve the continuity of care through strengthening the provider-patient relationships.

Evaluation of the National Notifiable Disease Surveillance System in Taiwan: an example of varicella reporting.

Despite the mandatory reporting by laws, the incompleteness of notifiable infectious disease reporting is well-documented in many countries for various diseases. The purpose of this study is to investigate the completeness of varicella reporting in Taiwan. Annual reports of National Notifiable Disease Surveillance System in Taiwan were compared to the annual outpatient claims of National Health Insurance (NHI) in the years of 2000, 2001, and 2002. Age and area-specific reporting rates of varicella were calculated by dividing the respective reported cases by the number of incidence cases. The reporting rate was the highest in aged 0 year in all years, followed by the 20-29- and 30-39-year groups. The reporting rate in each age group increased gradually during the study period. Other than Taipei City, the reporting rates in all regions were below 9% during this period. This study suggested that varicella reporting rate is very low in Taiwan. In addition, the reporting rates were inconsistent in 2000-2002, making the estimation of prevalence and vaccine efficacy using data from the National Notifiable Disease Surveillance System almost impossible. This study indicated that the physicians in Taiwan should improve their knowledge and attitude toward notifiable infectious diseases.

Development of a risk-adjusted capitation model based on principal inpatient diagnoses in Taiwan.

BACKGROUND AND PURPOSE: Taiwan's National Health Insurance (NHI) program has considered the use of capitation payments to health care providers as a method for control of the rising costs of the system. The establishment of capitation payments usually requires the performance of risk adjustment. The purposes of this study were to develop a diagnosis-based risk adjustment model for the NHI and to evaluate its predictability. METHODS: Using a 2% random sample of 371,620 NHI enrollees, the authors developed a Taiwan version of the Principal Inpatient Diagnosis Cost Groups (TPIPDCGs) from 1996 claim records to predict an individual's expenditure in 1997. Weighted least squares regression models were built in an estimation sample (two-thirds of the study sample), and were cross-validated in a validation sample (the remaining one-third of the study sample). Predictive R2 and predictive ratios were used to evaluate the model's predictability. RESULTS: Only 7.88% of the study sample could be classified into 1 of the 16 TPIPDCGs. Combined with demographic variables, which alone could explain 3.7% of the variation in an individual's future expenditure, the risk adjustment model based on TPIPDCGs could explain 12.2% of expenditure variation. In addition, the finding that the predictive ratios of the TPIPDCG model approximated unity better than those of the demographic model in all subgroups indicates that the capitation payment as predicted by the TPIPDCG model for each subgroup would better correlate to the actual spending. CONCLUSION: Taiwan's risk-adjusted capitation model based on principal inpatient diagnoses has higher predictability on individual's future expenditure than its counterpart in the USA. This finding provides insight into not only the development of Taiwan's diagnosis-based risk adjustment models but also the necessity of modification when applying foreign-developed risk adjustment models to the NHI.

Anti-4-1BB-based immunotherapy for autoimmune diabetes: lessons from a transgenic non-obese diabetic (NOD) model.

Various therapeutic strategies have been developed to tolerize autoreactive T cells and prevent autoimmune pathology in type 1 diabetes. 4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a costimulatory receptor primarily expressed on activated T cells. The administration of an agonistic anti-4-1BB antibody (2A) dramatically reduced the incidence and severity of experimental autoimmune encephalomyelitis (EAE). Treatment with the same antibody in Fas-deficient MRL/lpr mice blocked lymphadenopathy and lupus-like autoimmune processes. Paradoxically, transgenic non-obese diabetic (NOD) mice overexpressing membrane-bound agonistic single-chain anti-4-1BB Fv in pancreatic beta cells developed more severe diabetes than their non-transgenic littermates, with earlier onset, faster diabetic processes, and higher mortality. Forty percent of transgenic mice developed diabetes by 4 weeks of age, compared with their control littermates, which first exhibited diabetes at 14 weeks. The frequency of diabetes in female transgenics reached 70% by 8 weeks of age. Most female transgenic mice died around 12 weeks. Consistent with this, transgenic mice developed earlier and more severe insulitis and showed stronger GAD-specific T-cell responses, compared with age-matched control littermates. Our results indicate an adverse effect of transgenic anti-4-1BB scFv in NOD mice and suggest a potential risk of this anti-4-1BB-based immunotherapy for autoimmune diseases.

Healthcare utilization patterns and risk adjustment under Taiwan's National Health Insurance system.

BACKGROUND AND PURPOSE: Some recent proposals for reform of Taiwan's National Health Insurance (NHI) system include risk adjustment mechanisms. However, there is a paucity of research on risk adjustment and its utilization in health insurance systems in Taiwan. The purposes of this study were to determine the healthcare utilization pattern and to develop a risk assessment model for capitation payments under NHI. METHODS: The individual enrollment and medical expenditure data for 1996 and 1997 were obtained from the Bureau of NHI. A random sample of 360,037 beneficiaries was divided into two sub-samples: one for model building and one for validation. Linear regression was employed to estimate the relationship between each individual's 1997 total expenditure and risk adjusters, i.e., age, gender, prior years' medical spending, and catastrophic status. RESULTS: The 10- to 14-years age group had the lowest total expenditure of $NT 3,055 ($US 1 = $NT 27.5 in 1996) in 1996, while the 65 years and over age group had the highest at almost 10 times more than the lowest. The distributions of total expenditure for both genders followed the familiar J-shaped curve. The average of the total expenditure of individuals with a catastrophic diagnosis was more than 17 times that of individuals without. Age and gender resulted in a predictive R2 of only 3.8% in the risk assessment model. By including prior total expenditure, the predictive R2 increased to 24.2%. Further addition of catastrophic status increased the predictability slightly to 25%. Prior outpatient expenditure predicted 72% of subsequent outpatient expenditure, but prior inpatient expenditure predicted only 3% of subsequent inpatient expenditure. CONCLUSIONS: As in other countries, age and gender provided only limited predictability in risk assessment. On the other hand, prior outpatient expenditure in this study provided relatively superior predictability in risk assessment. Prudence is required when including prior utilization as a part of the risk assessment model in calculating capitation payments, as this may indirectly encourage unnecessary use of healthcare services.