Krüppel-like Factor 10 as a Prognostic and Predictive Biomarker of Radiotherapy in Pancreatic Adenocarcinoma.

The prognosis of pancreatic adenocarcinoma (PDAC) remains poor, with a 5-year survival rate of 12%. Although radiotherapy is effective for the locoregional control of PDAC, it does not have survival benefits compared with systemic chemotherapy. Most patients with localized PDAC develop distant metastasis shortly after diagnosis. Upfront chemotherapy has been suggested so that patients with localized PDAC with early distant metastasis do not have to undergo radical local therapy. Several potential tissue markers have been identified for selecting patients who may benefit from local radiotherapy, thereby prolonging their survival. This review summarizes these biomarkers including SMAD4, which is significantly associated with PDAC failure patterns and survival. In particular, Krüppel-like factor 10 (KLF10) is an early response transcription factor of transforming growth factor (TGF)-ß. Unlike TGF-ß in advanced cancers, KLF10 loss in two-thirds of patients with PDAC was associated with rapid distant metastasis and radioresistance; thus, KLF10 can serve as a predictive and therapeutic marker for PDAC. For patients with resectable PDAC, a combination of KLF10 and SMAD4 expression in tumor tissues may help select those who may benefit the most from additional radiotherapy. Future trials should consider upfront systemic therapy or include molecular biomarker-enriched patients without early distant metastasis.

Impact of pathological nodal staging and tumour differentiation on survival and postoperative radiotherapy in completely resected stage IIIA Non-small-cell lung cancer.

BACKGROUND: Tumour differentiation is an important index for adjuvant therapy in many cancers; however, non-small cell lung cancer (NSCLC) is an exception. Furthermore, postoperative radiotherapy (PORT) is controversial in patients with NSCLC with N0-1 and N2 disease. We aimed to evaluate the impact of tumour-related factors on overall survival (OS), cancer-specific survival (CSS), and distant control (DC) in patients with completely resected stage IIIA NSCLC. MATERIALS AND METHODS: Patients with stage IIIA non-metastatic NSCLC who underwent complete resection and adjuvant chemotherapy were identified from the Taiwan Cancer Registry (January 2007-December 2017). Logistic regression analysis was performed to determine the factors associated with PORT. Survival and relapse outcomes were compared using log-rank tests and Cox regression analysis. Sensitivity analysis was performed using propensity score-matched pairs. RESULTS: In total, 1,897 patients were included and stratified according to PORT use (PORT vs. non-PORT). After adjusting for covariates, PORT was not found to be associated with improved survival outcomes. In patients with poorly differentiated tumours and N2 disease, absolute benefits for OS (adjusted hazard ratio [aHR] 0.76), CSS (aHR 0.80), and DC (aHR 0.74) were observed. Multivariable hazard models of propensity score-matched pN2 disease and poorly differentiated tumour subgroups also showed significant survival benefit with PORT treatment. CONCLUSIONS: Patients with poorly differentiated tumours and receiving PORT for pN2 disease showed a lower risk of distant recurrence and more favourable survival outcomes in stage IIIA NSCLC with R0 resection.

Selection of patients with pancreatic adenocarcinoma who may benefit from radiotherapy.

Despite combination chemotherapy demonstrating a positive effect on survival, the clinical outcomes of pancreatic adenocarcinoma (PDAC) remain poor. Radiotherapy was previously a component of the curative treatment of PDAC. Advances in imaging and computer sciences have enabled the prescription of higher dosage of radiation focused on tumours with minimal toxicity to normal tissue. However, the role of radiotherapy has not been established in the curative treatment of localized PDAC because of the conflicting results from large prospective trials. Most studies have demonstrated improved locoregional control but no survival benefit from additional chemoradiotherapy (CRT) in addition to chemotherapy for resectable, borderline or locally advanced PDAC. The improved locoregional control enabled by CRT does not cause extended survival because of rapid distant progression in a significant proportion of patients with PDAC. Several single-institute studies of prescribing intensive chemotherapy with modern ablative radiotherapy for locally advanced PDAC have demonstrated extended survival with an acceptable safety profile. In an analysis after long-term follow-up, the PREOPANC study demonstrated a survival benefit from neoadjuvant gemcitabine-based CRT in resected PDAC relative to upfront surgery followed by adjuvant gemcitabine only. These observations indicated that the role of radiotherapy in PDAC should be evaluated in a subgroup of patients without rapid distant progression because systemic therapy for PDAC remains underdeveloped. We reviewed critical imaging, tissue, liquid and clinical biomarkers to differentiate the heterogeneous biologic spectra of patients with PDAC to identify those who may benefit the most from local radiotherapy. Exclusion of patients with localised PDAC who develop distant progression in a short time and undergo extended upfront chemotherapy for over 4 months may enable the identification of a survival benefit of local radiotherapy. Though promising, the effectiveness of biomarkers must be validated in a multi-institutional prospective study of patients with PDAC receiving CRT or not receiving CRT.

Krüppel-like factor 10 modulates stem cell phenotypes of pancreatic adenocarcinoma by transcriptionally regulating notch receptors.

BACKGROUND: Pancreatic adenocarcinoma (PDAC) is well known for its rapid distant metastasis and local destructive behavior. Loss of Krüppel-like factor 10 (KLF10) contributes to distant migration of PDAC. The role of KLF10 in modulating tumorigenesis and stem cell phenotypes of PDAC is unclear. METHODS: Additional depletion of KLF10 in KC (LSL: KrasG12D; Pdx1-Cre) mice, a spontaneous murine PDAC model, was established to evaluate tumorigenesis. Tumor specimens of PDAC patients were immune-stained of KLF10 to correlate with local recurrence after curative resection. Conditional overexpressing KLF10 in MiaPaCa and stably depleting KLF10 in Panc-1 (Panc-1-pLKO-shKLF10) cells were established for evaluating sphere formation, stem cell markers expression and tumor growth. The signal pathways modulated by KLF10 for PDAC stem cell phenotypes were disclosed by microarray analysis and validated by western blot, qRT-PCR, luciferase reporter assay. Candidate targets to reverse PDAC tumor growth were demonstrated in murine model. RESULTS: KLF10, deficient in two-thirds of 105 patients with resected pancreatic PDAC, was associated with rapid local recurrence and large tumor size. Additional KLF10 depletion in KC mice accelerated progression from pancreatic intraepithelial neoplasia to PDAC. Increased sphere formation, expression of stem cell markers, and tumor growth were observed in Panc-1-pLKO-shKLF10 compared with vector control. Genetically or pharmacologically overexpression of KLF10 reversed the stem cell phenotypes induced by KLF10 depletion. Ingenuity pathway analysis and gene set enrichment analysis showed that Notch signaling molecules, including Notch receptors 3 and 4, were over-expressed in Panc-1-pLKO-shKLF10. KLF10 transcriptionally suppressed Notch-3 and -4 by competing with E74-like ETS transcription factor 3, a positive regulator, for promoter binding. Downregulation of Notch signaling, either genetically or pharmacologically, ameliorated the stem cell phenotypes of Panc-1-pLKO-shKLF10. The combination of metformin, which upregulated KLF10 expression via phosphorylating AMPK, and evodiamine, a non-toxic Notch-3 methylation stimulator, delayed tumor growth of PDAC with KLF10 deficiency in mice without prominent toxicity. CONCLUSIONS: These results demonstrated a novel signaling pathway by which KLF10 modulates stem cell phenotypes in PDAC through transcriptionally regulating Notch signaling pathway. The elevation of KLF10 and suppression of Notch signaling may jointly reduce PDAC tumorigenesis and malignant progression.

Carbohydrate Antigen 19-9 Response to Initial Adjuvant Chemotherapy Predicts Survival and Failure Pattern of Resected Pancreatic Adenocarcinoma but Not Which Patients Are Suited for Additional Adjuvant Chemoradiation Therapy: From a Prospective Randomized Study.

PURPOSE: The predictive value of carbohydrate antigen 19-9 (CA19-9) for adjuvant chemo(radiation) therapy of resected pancreatic adenocarcinoma (PDAC) is undefined. METHODS AND MATERIALS: We analyzed CA19-9 levels in patients with resected PDAC in a prospective randomized trial of adjuvant chemotherapy with or without additional chemoradiation therapy (CRT). Patients with postoperative CA19-9 ≤92.5 U/mL and serum bilirubin ≤2 mg/dL were randomized to 2 arms: patients in 1 arm received 6 cycles of gemcitabine, whereas those in the other received 3 cycles of gemcitabine followed by CRT and another 3 cycles of gemcitabine. Serum CA19-9 was measured every 12 weeks. Those who had CA19-9 levels always <3 U/mL were excluded from the exploratory analysis. RESULTS: One hundred forty-seven patients were enrolled in this randomized trial. Twenty-two patients with CA19-9 levels always ≤3 U/mL were excluded from the analysis. For the 125 participants, median overall survival (OS) and recurrence-free survival were 23.1 and 12.1 months, respectively, with no significant differences between the study arms. Postresection CA19-9 levels and, to a lesser extent, CA19-9 change predicted OS (P = .040 and .077, respectively). For the 89 patients who completed the initial 3 cycles of adjuvant gemcitabine, the CA19-9 response was significantly correlated with initial failure over the distant site (P = .023) and OS (P = .0022). Despite a trend of less initial failure over the locoregional area (P = .031), neither postoperative CA19-9 level nor CA19-9 response helped to select patients who might have a survival benefit from additional adjuvant CRT. CONCLUSIONS: CA19-9 response to initial adjuvant gemcitabine predicts survival and distant failure of PDAC after resection; however, it cannot select patients suited for additional adjuvant CRT. Monitoring CA19-9 levels during adjuvant therapy for postoperative patients with PDAC may guide therapeutic decisions to prevent distant failure.

A phase II randomised trial of induction chemotherapy followed by concurrent chemoradiotherapy in locally advanced pancreatic cancer: the Taiwan Cooperative Oncology Group T2212 study.

BACKGROUND: The objective of this study was to evaluate the efficacy and safety of induction chemotherapy (ICT), GOFL (gemcitabine, oxaliplatin plus fluorouracil (5-FU)/leucovorin) versus modified FOLFIRINOX (irinotecan, oxaliplatin plus 5-FU/leucovorin), followed by concurrent chemoradiotherapy (CCRT) in locally advanced pancreatic adenocarcinoma (LAPC). METHODS: Chemo-naive patients with measurable LAPC were eligible and randomly assigned to receive biweekly ICT with either mFOLFIRINOX or GOFL for 3 months. Patients without systemic progression would have 5-FU- or gemcitabine-based CCRT (5040 cGy/28 fractions) and were then subjected to surgery or continuation of chemotherapy until treatment failure. The primary endpoint was 9-month progression-free survival (PFS) rate. RESULTS: Between July 2013 and January 2019, 55 patients were enrolled. After ICT, 21 (77.8%) of 27 patients who received mFOLFIRINOX and 17 (60.7%) of 28 patients who received GOFL completed CCRT. Of them, one and five had per-protocol R0/R1 resection. On intent-to-treat analysis, the 9-month PFS rate, median PFS and overall survival in mFOLFIRINOX and GOFL arms were 30.5% versus 35.9%, 6.6 (95% confidence interval: 5.9-12.5) versus 7.6 months (3.9-12.3) and 19.6 (13.4-22.9) versus 17.9 months (13.4-23.9), respectively. Grade 3-4 neutropenia and diarrhoea during induction mFOLFIRINOX and GOFL were 37.0% versus 21.4% and 14.8% versus 3.6%, respectively. CONCLUSION: Induction GOFL and mFOLFIRINOX followed by CCRT provided similar clinical outcomes in LAPC patients. GOV IDENTIFIER: NCT01867892.

Upregulating sirtuin 6 ameliorates glycolysis, EMT and distant metastasis of pancreatic adenocarcinoma with krüppel-like factor 10 deficiency.

Krüppel-like factor 10 (KLF10) is a tumor suppressor in multiple cancers. In a murine model of spontaneous pancreatic adenocarcinoma (PDAC), additional KLF10 depletion accelerated distant metastasis. However, Klf10 knockout mice, which suffer from metabolic disorders, do not develop malignancy. The mechanisms of KLF10 in PDAC progression deserve further exploration. KLF10-depleted and KLF10-overexpressing PDAC cells were established to measure epithelial-mesenchymal transition (EMT), glycolysis, and migration ability. A murine model was established to evaluate the benefit of genetic or pharmacological manipulation in KLF10-depleted PDAC cells (PDACshKLF10). Correlations of KLF10 deficiency with rapid metastasis, elevated EMT, and glycolysis were demonstrated in resected PDAC tissues, in vitro assays, and murine models. We identified sirtuin 6 (SIRT6) as an essential mediator of KLF10 that modulates EMT and glucose homeostasis. Overexpressing SIRT6 reversed the migratory and glycolytic phenotypes of PDACshKLF10 cells. Linoleic acid, a polyunsaturated essential fatty acid, upregulated SIRT6 and prolonged the survival of mice injected with PDACshKLF10. Modulating HIF1α and NFκB revealed that EMT and glycolysis in PDAC cells were coordinately regulated upstream by KLF10/SIRT6 signaling. Our study demonstrated a novel KLF10/SIRT6 pathway that modulated EMT and glycolysis coordinately via NFκB and HIF1α. Activation of KLF10/SIRT6 signaling ameliorated the distant progression of PDAC.Clinical Trial Registration: ClinicalTrials.gov. identifier: NCT01666184.

High expression of krüppel-like factor 10 or Smad4 predicts clinical benefit of adjuvant chemoradiotherapy in curatively resected pancreatic adenocarcinoma: From a randomized phase III trial.

PURPOSE: Our previous studies have demonstrated that Krüppel-like factor 10 (Klf10) modulated tumor radiation resistance and helps to predict clinical outcomes of pancreatic adenocarcinoma (PDAC). This study aimed to evaluate whether the expression levels of Klf10, Smad4 and Runx3 can help predict the benefits of adjuvant chemoradiotherapy (CRT) in resected PDAC. METHODS AND MATERIALS: Tissue specimens were collected from 111 patients with curatively resected PDAC who were enrolled into a randomized trial comparing adjuvant gemcitabine with or without CRT. Immunohistochemical expression of biomarkers was quantified by pathologists blinded to patient outcomes through a grading system based on the extent and intensity of staining. The predictive value of biomarkers was analyzed using SAS statistical software. RESULTS: In total, 56 and 55 patients received adjuvant gemcitabine alone and additional CRT, respectively. The expression levels of Klf10, Smad4 and postoperative CA19-9 were significantly correlated with overall survival (OS) (p = 0.013, 0.045, and 0.047, respectively). Multivariable analysis showed that the expression level of postoperative serum CA19-9 and tumor tissue Klf10 expression level were significant predictors for OS (p = 0.038, and 0.028, respectively). Patients with high Klf10 or Smad4 (n = 55), had a significantly better local recurrence-free survival (∞ vs 19.8 months; p = 0.026) and a longer OS (33.0 vs 23.0 months; p = 0.12) if they received additional adjuvant CRT than gemcitabine only. The results were similar after adjusted by postoperative level of CA19-9. CONCLUSION: Patients with curatively resected PDAC and a high expression of either Klf10 or Smad4 have high chances of benefiting from adjuvant CRT. Combining Klf10 and Smad4 to predict the benefits of adjuvant CRT in resected PDAC deserves further validation.

Therapeutic Effect of Repurposed Temsirolimus in Lung Adenocarcinoma Model.

Lung cancer is one of the major cause of cancer-related deaths worldwide. The poor prognosis and resistance to both radiation and chemotherapy urged the development of potential targets for lung cancer treatment. In this study, using a network-based cellular signature bioinformatics approach, we repurposed a clinically approved mTOR inhibitor for renal cell carcinomans, temsirolimus, as the potential therapeutic candidate for lung adenocarcinoma. The PI3K-AKT-mTOR pathway is known as one of the most frequently dysregulated pathway in cancers, including non-small-cell lung cancer. By using a well-documented lung adenocarcinoma mouse model of human pathophysiology, we examined the effect of temsirolimus on the growth of lung adenocarcinoma in vitro and in vivo. In addition, temsirolimus combined with reduced doses of cisplatin and gemcitabine significantly inhibited the lung tumor growth in the lung adenocarcinoma mouse model compared with the temsirolimus alone or the conventional cisplatin-gemcitabine combination. Functional imaging techniques and microscopic analyses were used to reveal the response mechanisms. Extensive immunohistochemical analyses were used to demonstrate the apparent effects of combined treatments on tumor architecture, vasculature, apoptosis, and the mTOR-pathway. The present findings urge the further exploration of temsirolimus in combination with chemotherapy for treating lung adenocarcinoma.

The Role of Consolidation Chemoradiotherapy in Locally Advanced Pancreatic Cancer Receiving Chemotherapy: An Updated Systematic Review and Meta-Analysis.

PURPOSE: The role of consolidation chemoradiation (CCRT) after systemic chemotherapy in locally advanced pancreatic cancer (LAPC) is still controversial. We aim to evaluate the effectiveness of CCRT in LAPC using systematic review and meta-analysis of prospective studies. MATERIALS AND METHODS: Prospective clinical trials of LAPC receiving chemotherapy with or without subsequent CCRT were included in the analysis. We systematically searched in PubMed, MEDLINE, Embase, and Web of Science. The primary outcome of interest was 1-year survival. Secondary endpoints were median overall survival, progression-free survival, toxicity, and resection rate. RESULTS: Forty-one studies with 49 study arms were included with a total of 1,018 patients receiving CCRT after induction chemotherapy (ICT) and 954 patients receiving chemotherapy alone. CCRT after ICT did not improve 1-year survival significantly in LAPC patients compared with chemotherapy alone (58% vs. 52%). ICT lasted for at least 3 months revealed significantly improved survival of additional CCRT to LAPC patients compared to chemotherapy alone (65% vs. 52%). A marginal survival benefit of consolidation CCRT was noted in studies using maintenance chemotherapy (59% vs. 52%), and fluorouracil-based CCRT (64% vs. 52%), as well as in studies conducted after the 2010 (64% vs. 55%). CONCLUSION: The survival benefit of ICT+CCRT over chemotherapy alone in treating LAPC was noted when ICT lasted for at least 3 months. Fluorouracil-based CCRT, and maintenance chemotherapy were associated with improved clinical outcomes.

Characterization of a transgenic mouse model exhibiting spontaneous lung adenocarcinomas with a metastatic phenotype.

Developing lung cancer in mouse models that display similarities of both phenotype and genotype will undoubtedly provide further and better insights into lung tumor biology. Moreover, a high degree of pathophysiological similarity between lung tumors from mouse models and their human counterparts will make it possible to use these mouse models for preclinical tests. Ovine pulmonary adenocarcinomas (OPAs) present the same symptoms as adenocarcinomas in humans and are caused by a betaretrovirus. OPAs have served as an exquisite model of carcinogenesis for human lung adenocarcinomas. In this study, we characterized the histopathology and transcriptome profiles of a jaagsiekte sheep retrovirus (JSRV)-envelope protein (Env) transgenic mouse model with spontaneous lung tumors, and associations of the transcriptome profiles with tumor invasion/metastasis, especially the phenomenon of the epithelial-mesenchymal transition (EMT). Genetic information obtained from an expression array was analyzed using an ingenuity pathways analysis (IPA) and human disease database (MalaCards). By careful examination, several novel EMT-related genes were identified from tumor cells using RT-qPCR, and these genes also scored high in MalaCards. We concluded that the JSRV-Env mouse model could serve as a spontaneous lung adenocarcinoma model with a metastatic phenotype, which will benefit the study of early-onset and progression of lung adenocarcinoma. In addition, it can also be a valuable tool for biomarkers and drug screening, which will be helpful in developing intervention therapies.

Krüpple-like factor 10 regulates radio-sensitivity of pancreatic cancer via UV radiation resistance-associated gene.

BACKGROUND AND PURPOSE: Krüpple-like factor 10 (Klf10), an early response gene of TGFß, was reported to be a prognostic biomarker for pancreatic cancer survival. The role of Klf10 in predicting tumor response to cancer treatment is unknown. MATERIALS AND METHODS: Genetically manipulated MiaPaCa and Panc-1 cells were established to evaluate clonogenic survival, autophagy, apoptosis and DNA repair after radiation. The interaction between Klf10 and UV radiation resistance-associated gene (UVRAG) was demonstrated by ChiP-PCR and luciferase reporter assay. Orthotopic murine tumor model and clinical specimens were used to evaluate radio-sensitivity of pancreatic cancer. RESULTS: We found Klf10 silencing correlates with enhanced pancreatic cancer clonogenic survival and murine tumor growth after radiation. UVRAG was an essential down-stream mediator transcriptionally suppressed by Klf10. Silencing UVRAG mRNA in Klf10 depleted Panc-1 cells reversed the radio-resistant phenotypes including decreased apoptosis and enhanced DNA repair as well as autophagy. Metformin, an anti-diabetic agent, was found to increase Klf10 and suppress UVRAG expression to improve radiation cytotoxicity in pancreatic cancer. The predictive value of Klf10 in radiation response and the inverse correlation with UVRAG were confirmed in cohorts of pancreatic cancer patients. CONCLUSIONS: Klf10 is a potential biomarker in predicting and sensitizing radiation effect in pancreatic cancer.

A phase I clinical study of immunotherapy for advanced colorectal cancers using carcinoembryonic antigen-pulsed dendritic cells mixed with tetanus toxoid and subsequent IL-2 treatment.

BACKGROUND: To better evaluate and improve the efficacy of dendritic cell (DC)-based cancer immunotherapy, we conducted a clinical study of patients with advanced colorectal cancer using carcinoembryonic antigen (CEA)-pulsed DCs mixed with tetanus toxoid and subsequent interleukin-2 treatment. The tetanus toxoid in the vaccine preparation serves as an adjuvant and provides a non-tumor specific immune response to enhance vaccine efficacy. The aims of this study were to (1) evaluate the toxicity of this treatment, (2) observe the clinical responses of vaccinated patients, and (3) investigate the immune responses of patients against CEA before and after treatment. METHODS: Twelve patients were recruited and treated in this phase I clinical study. These patients all had metastatic colorectal cancer and failed standard chemotherapy. We first subcutaneously immunized patients with metastatic colorectal cancer with 1 × 10(6) CEA-pulsed DCs mixed with tetanus toxoid as an adjuvant. Patients received 3 successive injections with 1 × 10(6) CEA-pulsed DCs alone. Low-dose interleukin-2 was administered subcutaneously following the final DC vaccination to boost the growth of T cells. Patients were evaluated for adverse event and clinical status. Blood samples collected before, during, and after treatment were analyzed for T cell proliferation responses against CEA. RESULTS: No severe treatment-related side effects or toxicity was observed in patients who received the regular 4 DC vaccine injections. Two patients had stable disease and 10 patients showed disease progression. A statistically significant increase in proliferation against CEA by T cells collected after vaccination was observed in 2 of 9 patients. CONCLUSIONS: The results of this study indicate that it is feasible and safe to treat colorectal cancer patients using this protocol. An increase in the anti-CEA immune response and a clinical benefit was observed in a small fraction of patients. This treatment protocol should be further evaluated in additional colorectal cancer patients with modifications to enhance T cell responses. TRIAL REGISTRATION: ClinicalTrials.gov (identifier NCT00154713 ), September 8, 2005.

Bone marrow derived macrophages fuse with intestine stromal cells and contribute to chronic fibrosis after radiation.

BACKGROUND AND PURPOSE: Bone marrow-derived cells (BMDC) have been demonstrated to play a critical role in intestine regeneration. However, organ fibrosis was one of the major side effects of bone marrow (BM) transplantation. It warrants further investigation on the mechanisms of BM cell therapy in radiation induced intestine damage. MATERIALS AND METHODS: We established three murine models to evaluate BMDC within intestines after radiation, including cre-loxP system of transgenic mice. In vitro co-culture between murine BM with human intestine stromal cells was also performed to measure the level of fusion and fibrosis after treatment with anti-fibrotic agents or after macrophage depletion. RESULTS: Despite complete recovery of epithelial mucosa from radiation damage, we found persistent proliferation and repopulation of BMDC within the lamina propria. Fusion between BM derived monocytic and intestine stromal cells correlated with the level of fibrosis and proliferation index. Depleting macrophages genetically using CD11b-DTR mouse model or pharmacologically using clodronate liposome reduced the level of cell fusion and intestine fibrosis. CONCLUSIONS: Fibrotic cues from intestine enhance fusion between BM-derived monocytes/macrophages with intestine stromal cells. The fusion hybrids promote cell cycle re-entry, proliferation and reinforce fibrosis signal. Depleting macrophages interferes with cell fusion and ameliorates radiation-induced intestine fibrosis.

Optimal combination of antiangiogenic therapy for hepatocellular carcinoma.

The success of sorafenib in prolonging survival of patients with hepatocellular carcinoma (HCC) makes therapeutic inhibition of angiogenesis a component of treatment for HCC. To enhance therapeutic efficacy, overcome drug resistance and reduce toxicity, combination of antiangiogenic agents with chemotherapy, radiotherapy or other targeted agents were evaluated. Nevertheless, the use of antiangiogenic therapy remains suboptimal regarding dosage, schedule and duration of therapy. The issue is further complicated by combination antiangiogenesis to other cytotoxic or biologic agents. There is no way to determine which patients are most likely respond to a given form of antiangiogenic therapy. Activation of alternative pathways associated with disease progression in patients undergoing antiangiogenic therapy has also been recognized. There is increasing importance in identifying, validating and standardizing potential response biomarkers for antiangiogenesis therapy for HCC patients. In this review, biomarkers for antiangiogenesis therapy including systemic, circulating, tissue and imaging ones are summarized. The strength and deficit of circulating and imaging biomarkers were further demonstrated by a series of studies in HCC patients receiving radiotherapy with or without thalidomide.

Perfusion parameters of dynamic contrast-enhanced magnetic resonance imaging predict outcomes of hepatocellular carcinoma receiving radiotherapy with or without thalidomide.

BACKGROUND: To correlate between signal parameters using dynamic contrast-enhanced magnetic resonance imaging (DCEMRI) and outcomes of hepatocellular carcinoma (HCC) receiving radiotherapy with or without concomitant thalidomide. METHODS: DCEMRI was performed in advanced HCC patients undergoing radiotherapy with or without concomitant thalidomide. Initial first-pass enhancement slopes (slope) and peak enhancement ratios (peak) were measured over an operator-defined region of interest over tumor and non-tumor liver parenchyma. The perfusion parameters were correlated with clinical outcomes. The study was registered with ClinicalTrials.gov. (identifier NCT00155272). RESULTS: Forty-three patients were evaluable. There were 18 partial responses (PRs), 5 minimal responses (MRs), 17 stable diseases (SDs), and 3 progressive diseases (PDs). Baseline perfusion parameters as well as slope at 14 days of radiotherapy were higher in patients with PR or MR compared to SD or PD (0.81 ± 0.29 vs. 0.49 ± 0.34, p < 0.01; 0.39 ± 0.15 vs. 0.28 ± 0.16, p = 0.02; 0.97 ± 0.38 vs. 0.46 ± 0.26, p < 0.01; respectively). Multivariate analysis revealed perfusion parameters over liver parenchyma, but not over tumor, and independently predicted progression-free and overall survival (182 ± 33 vs. 105 ± 26 days, p = 0.01; 397 ± 111 vs. 233 ± 19 days, p = 0.001 respectively). For 22 patients receiving concomitant thalidomide, the perfusion parameters were not significantly different from those receiving radiotherapy alone. CONCLUSIONS: Signal parameters of DCEMRI over tumor and liver parenchyma correlated with tumor response and survival, respectively, in HCC patients receiving radiotherapy.

Circulating cells contribute to cardiomyocyte regeneration after injury.

RATIONALE: The contribution of bone marrow-borne hematopoietic cells to the ischemic myocardium has been documented. However, a pivotal study reported no evidence of myocardial regeneration from hematopoietic-derived cells. The study did not take into account the possible effect of early injury-induced signaling as the test mice were parabiotically paired to partners immediately after surgery-induced myocardial injury when cross-circulation has not yet developed. OBJECTIVE: To re-evaluate the role of circulating cells in the injured myocardium. METHODS AND RESULTS: By combining pulse-chase labeling and parabiosis model, we show that circulating cells derived from the parabiont expressed cardiac-specific markers in the injured myocardium. Genetic fate mapping also revealed that circulating hematopoietic cells acquired cardiac cell fate by means of cell fusion and transdifferentiation. CONCLUSIONS: These results suggest that circulating cells participate in cardiomyocyte regeneration in a mouse model of parabiosis when the circulatory system is fully developed before surgery-induced heart injury.

Radiation sensitization of tumor cells induced by shear stress: the roles of integrins and FAK.

Recent studies revealed that the interstitial fluid flow in and around tumor tissue not only played an important role in delivering anticancer agents, but also affected the microenvironment, mostly hypoxia, in modulating tumor radio-sensitivity. The current study investigated the hypoxia-independent mechanisms of flow-induced shear stress in sensitizing tumors to radiation. Colon cancer cells were seeded onto glass slides pre-coated with fibronectin. A parallel-plate flow chamber system was used to impose fluid shear stress. Cell proliferation, apoptosis and colony assays were measured after shear stress and/or radiation. Cell cycle analysis and immunoblots of cell adhesion signal molecules were evaluated. The effect of shear stress was reversed by modulating integrin ß1 or FAK. Shear stress of 12dyne/cm(2) for 24h, but not 3h, enhanced the radiation induced cytotoxicity to colon cancer cells. Protein expression of FAK was significantly down-regulated but not transcriptionally suppressed. By modulating integrin ß1 and FAK expression, we demonstrated that shear stress enhanced tumor radiosensitivity by regulating integrin ß1/FAK/Akt as well as integrin ß1/FAK/cortactin pathways. Shear stress in combination with radiation might regulate integrins signaling by recruiting and activating caspases 3/8 for FAK cleavage followed by ubiquitin-mediated proteasomal degradation. Shear stress enhanced the radiation toxicity to colon cancer cells through suppression of integrin signaling and protein degradation of FAK. The results of our study provide a strong rationale for cancer treatment that combines between radiation and strategy in modulating tumor interstitial fluid flow.

Destabilization of KLF10, a tumor suppressor, relies on thr93 phosphorylation and isomerase association.

KLF10 is now classified as a member of the Krüppel-like transcription factor family and acts as a tumor suppressor. Although KLF10 is originally named as TGF-ß-inducible early gene-1 and mimicking the anti-proliferative effect of TGF-ß in various carcinoma cells, the transcriptional upregulatory function of KLF10 has been described for a variety of cytokines and in many diseases. Through in vivo and in vitro phosphorylation assays, we identified that KLF10 is a phosphorylated protein in cells. Using yeast-two hybrid screening and site direct mutagenesis, we also identified PIN1 as a novel KLF10 associated protein. PIN1 is a peptidyl-prolyl isomerase enzyme belonging to the parvulin family, which specifically recognizes phosphorylated Ser/Thr-Pro containing substrates. Through protein-protein interaction assays, we showed that the Pro-directed Ser/Thr-Pro motif at Thr-93 in the KLF10 N-terminal region is essential for the interaction between KLF10 and PIN1. More importantly, PIN1 interacts with KLF10 in a phosphorylation-dependent manner and this interaction promotes KLF10 protein degradation in cells. Therefore, KLF10 shows shorter protein stability compared with mutant KLF10 that lacks PIN1 binding ability after cycloheximide treatments. The reversely correlated expression profile between KLF10 and PIN1 as observed in cell lines was also shown in clinic pancreatic cancer specimen. Using in vitro kinase assays and depletion assays, we were able to show that RAF-1 phosphorylates the Thr-93 of KLF10 and affects the KLF10 expression level in cells. Thus these findings as a whole indicate that RAF-1 phosphorylation and PIN1 isomerization together regulate KLF10 stability and further affect the role of KLF10 in tumor progression.

Bone marrow transplantation rescues intestinal mucosa after whole body radiation via paracrine mechanisms.

PURPOSE: Our previous study reveals bone marrow transplantation (BMT) recruits host marrow-derived myelomonocytic cells to radiation-injured intestine, enhancing stromal proliferation, leading secondarily to epithelial regeneration. In this study, we propose BMT ameliorates intestinal damage via paracrine mechanisms. MATERIALS AND METHODS: Angiogenic cytokines within the intestinal mucosa of mice after whole body irradiation (WBI) with or without BMT were measured by cytokine array and ELISA. BM conditioned medium (BMCM) with or without treatment with neutralizing antibodies to angiogenic cytokines were continuously infused into mice for three days after radiation. Carrageenan was used to deplete myelomonocytic cells of mice. RESULTS: BMT increased VEGF, bFGF and other angiogenic and chemotactic cytokines in the intestinal mucosa within 24h after WBI. Infusion of BMCM ameliorated radiation-induced intestinal damage with improved stromal activity and prolonged survival of mice. Neutralization of bFGF, PDGF and other angiogenic cytokines within BMCM abolished the mitigating effect to the intestine. Pretreatment of carrageenan to recipient mice reversed some of the cytokine levels, including VEGF, bFGF and IGF within the intestinal mucosa after BMT. CONCLUSIONS: Our result suggests BMT recruits host myelomonocytic cells and enhances intestinal stroma proliferation after radiation by secreting cytokines enhancing angiogenesis and chemotaxis. Host myelomonocytic cells further uplift the paracrine effect to enhance intestinal mucosal recovery.