Jason Liao

Targeting of survivin by nanoliposomal ceramide induces complete remission in a rat model of NK-LGL leukemia

The natural killer (NK) type of aggressive large granular lymphocytic (LGL) leukemia is a fatal illness that pursues a rapid clinical course. There are no effective therapies for this illness, and pathogenetic mechanisms remain undefined. Here we report that the survivin was highly expressed in both aggressive and chronic leukemic NK cells but not in normal NK cells. In vitro treatment of human and rat NK-LGL leukemia cells with cell-permeable, short-chain C₆-ceramide (C₆) in nanoliposomal formulation led to caspase-dependent apoptosis and diminished survivin protein expression, in a time- and dose-dependent manner. Importantly, systemic intravenous delivery of nanoliposomal ceramide induced complete remission in the syngeneic Fischer F344 rat model of aggressive NK-LGL leukemia. Therapeutic efficacy was associated with decreased expression of survivin in vivo. These data suggest that in vivo targeting of survivin through delivery of nanoliposomal C₆-ceramide may be a promising therapeutic approach for a fatal leukemia.

C6-ceramide nanoliposomes target the Warburg effect in chronic lymphocytic leukemia.

Ceramide is a sphingolipid metabolite that induces cancer cell death. When C6-ceramide is encapsulated in a nanoliposome bilayer formulation, cell death is selectively induced in tumor models. However, the mechanism underlying this selectivity is unknown. As most tumors exhibit a preferential switch to glycolysis, as described in the “Warburg effect”, we hypothesize that ceramide nanoliposomes selectively target this glycolytic pathway in cancer. We utilize chronic lymphocytic leukemia (CLL) as a cancer model, which has an increased dependency on glycolysis. In CLL cells, we demonstrate that C6-ceramide nanoliposomes, but not control nanoliposomes, induce caspase 3/7-independent necrotic cell death. Nanoliposomal ceramide inhibits both the RNA and protein expression of GAPDH, an enzyme in the glycolytic pathway, which is overexpressed in CLL. To confirm that ceramide targets GAPDH, we demonstrate that downregulation of GAPDH potentiates the decrease in ATP after ceramide treatment and exogenous pyruvate treatment as well as GAPDH overexpression partially rescues ceramide-induced necrosis. Finally, an in vivo murine model of CLL shows that nanoliposomal C6-ceramide treatment elicits tumor regression, concomitant with GAPDH downregulation. We conclude that selective inhibition of the glycolytic pathway in CLL cells with nanoliposomal C6-ceramide could potentially be an effective therapy for leukemia by targeting the Warburg effect.

miR-200b restoration and DNA methyltransferase inhibitor block lung metastasis of mesenchymal-phenotype hepatocellular carcinoma

Epithelial-to-mesenchymal transition (EMT) is associated with poor prognosis and metastasis in hepatocellular carcinoma. We have previously demonstrated an in vivo model of liver cancer in which mesenchymal cells post-EMT demonstrate a high rate of invasive growth and metastasis. Here, we investigate the role of microRNA 200 (miR-200) family members and epigenetic modifications on the maintenance of mesenchymal/metastatic phenotype after EMT. Mesenchymal cells post-EMT demonstrates high levels of E-box repressors Zeb1 and Zeb2 and downregulation of four miR-200 family members (miR-200a, miR-200b, miR-200c and miR-429). In addition, DNA sequencing after bisulfite modification demonstrates that several CpG sites within the E-cadherin promoter are methylated in mesenchymal cells. In mesenchymal cells, forced expression of miR-200b results in a significant increase in E-cadherin and a reduction in cell migration/invasion. Despite these mesenchymal-to-epithelial transition (MET) changes in vitro, there is no significant change in metastatic potential after miR-200b upregulation in vivo. After the mesenchymal cells were treated with combination of DNA methyltransferase (DNMT) inhibitor and upregulation of miR-200b, invasive phenotype was significantly reduced and metastatic potential was eliminated. Direct targeting of E-cadherin with short hairpin RNA does not restore metastatic potential after DNMT inhibition and miR-200b re-expression. In addition, restoration of E-cadherin alone was unable to block metastatic potential in primary mesenchymal cells. In conclusion, targeting mesenchymal liver cancer cells with miR-200b and DNMT inhibitor reduces metastatic potential irrespective of E-cadherin expression. Thus, the broader differentiation and MET effects of DNMT inhibition and miR-200b must be considered in terms of rescuing metastatic potential.

Characteristics of Incidentally Discovered Thyroid Cancer

IMPORTANCE The incidence of thyroid cancer has been steadily increasing; however, no clear reason for the increase in incidence has been identified. OBJECTIVES To compare incidentally discovered (ID) thyroid cancer via non-thyroid-related imaging with nonincidentally discovered (NID) thyroid cancer, as well as determine if differences in tumor characteristics and patient presentation in ID thyroid cancer may help elucidate the increasing incidence of this disease. DESIGN, SETTING, AND PARTICIPANTS Retrospective medical record review at an academic tertiary care medical center of 31 patients with ID thyroid cancer and 207 patients with NID thyroid cancer evaluated at our institution during a 12-month period. MAIN OUTCOMES AND MEASURES Patient demographics, tumor pathology, stage, tumor size, invasion, and metastasis were recorded. RESULTS Mean age at diagnosis was 56.4 years for the ID group and 41.8 years for the NID group (P < .001). The ID group was 54.8% male compared with 13.5% in the NID group (P < .001). The ID group had higher stage disease compared with the NID group (P = .003). There was no difference in tumor size (P = .91), invasion (P = .76), lymph node involvement, or distant metastases (P > .99). CONCLUSIONS AND RELEVANCE Patients with ID thyroid cancer tend to be older at presentation, have higher stage disease, and are more likely to be male compared with patients with NID thyroid cancer. There does not appear to be a significant difference in the size, pathology, or behavior of the tumor at presentation between ID and NID thyroid cancers. These findings imply that improved detection may not represent the only cause of the increased incidence of thyroid cancer.

Acid ceramidase is upregulated in AML and represents a novel therapeutic target

There is an urgent unmet need for new therapeutics in acute myeloid leukemia (AML) as standard therapy has not changed in the past three decades and outcome remains poor for most patients. Sphingolipid dysregulation through decreased ceramide levels and elevated sphingosine 1-phosphate (S1P) promotes cancer cell growth and survival. Acid ceramidase (AC) catalyzes ceramide breakdown to sphingosine, the precursor for S1P. We report for the first time that AC is required for AML blast survival. Transcriptome analysis and enzymatic assay show that primary AML cells have high levels of AC expression and activity. Treatment of patient samples and cell lines with AC inhibitor LCL204 reduced viability and induced apoptosis. AC overexpression increased the expression of anti-apoptotic Mcl-1, significantly increased S1P and decreased ceramide. Conversely, LCL204 induced ceramide accumulation and decreased Mcl-1 through post-translational mechanisms. LCL204 treatment significantly increased overall survival of C57BL/6 mice engrafted with leukemic C1498 cells and significantly decreased leukemic burden in NSG mice engrafted with primary human AML cells. Collectively, these studies demonstrate that AC plays a critical role in AML survival through regulation of both sphingolipid levels and Mcl-1. We propose that AC warrants further exploration as a novel therapeutic target in AML.

Clinical, Sociodemographic, and Service Provider Determinants of Guideline Concordant Colorectal Cancer Care for Appalachian Residents

BACKGROUND Colorectal cancer represents a significant cause of morbidity and mortality, particularly in Appalachia where high mortality from colorectal cancer is more prevalent. Adherence to treatment guidelines leads to improved survival. This paper examines determinants of guideline concordance for colorectal cancer. METHODS Colorectal cancer patients diagnosed in 2006-2008 from 4 cancer registries (Kentucky, Ohio, Pennsylvania, and North Carolina) were linked to Medicare claims (2005-2009). Final sample size after exclusions was 2,932 stage I-III colon, and 184 stage III rectal cancer patients. The 3 measures of guideline concordance include adjuvant chemotherapy (stage III colon cancer, <80 years), ≥12 lymph nodes assessed (resected stage I-III colon cancer), and radiation therapy (stage III rectal cancer, <80 years). Bivariate and multivariate analyses with clinical, sociodemographic, and service provider covariates were estimated for each of the measures. RESULTS Rates of chemotherapy, lymph node assessment, and radiation were 62.9%, 66.3%, and 56.0%, respectively. Older patients had lower rates of chemotherapy and radiation. Five comorbidities were significantly associated with lower concordance in the bivariate analyses: myocardial infarction, congestive heart failure, respiratory diseases, dementia with chemotherapy, and diabetes with adequate lymph node assessment. Patients treated by hospitals with no Commission on Cancer (COC) designation or lower surgical volumes had lower odds of adequate lymph node assessment. CONCLUSIONS Clinical, sociodemographic, and service provider characteristics are significant determinants of the variation in guideline concordance rates of 3 colorectal cancer measures.

Influence of omega-3 fatty acids on Tamoxifen-induced suppression of rat mammary carcinogenesis

We report here a detailed time course study of the individual and combined chemopreventive effects of Tamoxifen (Tam) and a high fish oil (FO) diet on multiple histologic parameters of mammary carcinogenesis. Groups of female Sprague‐Dawley rats were injected ip with 1‐methyl‐1‐nitrosourea at 50 days of age and assigned to either a control diet (20% corn oil [CO]) or a FO‐rich diet (10% FO + 10% CO) in the presence and absence of Tam in the diet (0.6 ppm). Rats were sacrificed at weeks 4 (before palpable tumors), 8 and 12 (when ∼90% of control rats had palpable tumors). Our results demonstrate a major effect of Tam in inhibiting the development of early preneoplastic lesions. FO, while having a marginal protective effect of it own, enhanced the antitumor action of Tam on all histologic parameters of carcinogenesis, although the effects of the combination were not statistically different from those of Tam alone. The combination of FO and Tam was the only intervention that induced regression of established preneoplastic lesions. We also found that in contrast to plasma, only target tissue n‐3 fatty acids (FAs) levels correlated with select tissue biomarkers of carcinogenesis whose expression was altered in a manner predictive of a protective effect. Our results demonstrating the potentially superior chemopreventive efficacy of Tam and n‐3FA have important translational implications. Our data also emphasize the importance of local factors in affecting target tissue levels and biologic effects of n‐3FA.

Incidental Internal Mammary Lymph Nodes Visualized on Screening Breast MRI

OBJECTIVE The frequency of visualization and size of internal mammary lymph nodes in women undergoing high-risk screening breast MRI is unknown. When these nodes are discovered on staging MRI of newly diagnosed breast cancer patients, management could present a treatment dilemma because normal size criteria do not exist. The aim of this study was to establish the average size and frequency of internal mammary lymph nodes observed in asymptomatic high-risk women undergoing screening breast MRI. MATERIALS AND METHODS We conducted a retrospective review of 108 women at high risk for breast cancer who underwent screening breast MRI between January 2010 and January 2014. Patients with new or previous diagnosis of breast cancer, prior nonbreast malignancy affecting the thorax or mediastinum, or previous radiation to the thorax were excluded. The presence, diameter, laterality, intercostal space, relationship to the internal mammary vessels, age, morphology, and clinical history of internal mammary lymph nodes were recorded. RESULTS Internal mammary lymph nodes were visualized in 50 of 108 high-risk patients, with an average size of 4.5 mm (range [± SD], 2-9 ± 1.59 mm). In the 50 women who had internal mammary lymph nodes visible on MRI, an average of 1.4 nodes (range, 1-3 nodes) were present. Internal mammary lymph nodes were more frequently visualized on the left (p < 0.001), at the second and third intercostal spaces (p = 0.007), and medial to the internal mammary vessels (p < 0.001). CONCLUSION In this small cohort, 1-3 presumed normal internal mammary lymph nodes measuring 2-9 mm (mean diameter 4.5 mm) were detected in about half of asymptomatic high-risk women presenting for screening MRI of the breasts.

Clinico-pathological correlation of serial measurement of circulating tumor cells in 24 metastatic colorectal cancer patients receiving chemotherapy reveals interpatient heterogeneity correlated with CEA levels but independent of KRAS and BRAF mutation

Background: The Veridex CellSearch is an FDA-approved technology for enumerating circulating tumor cells in blood samples of metastatic colorectal cancer mCRC) patients and has prognostic value. It is important to understand how counts of circulating tumor cells (CTCs), which are advocated to be tools for “liquid biopsy” of tumors, correlate with clinical and pathologic variables of significance in these patients. In this study, we have attempted to make such correlations along with evaluating how CTC counts change during the course of chemotherapy. Patients and methods: Following an IRB-approved protocol, blood samples were collected from 24 patients with mCRC along with relevant clinico-pathological data. Blood was collected at defined time-points both prior to as well as during the course of treatment with combination chemotherapy, and CTC counts were enumerated from7.5 ml of blood. Results: Seventeen out of 24 patients with mCRC showed a CTC count of 2 or less cells in 7.5 ml of blood at base-line assessment before chemotherapy while 7 patients showed 3 or more cells in 7.5 ml of blood at that point. A correlation was found between high carcino-embryonic antigen (CEA) levels and high CTC counts (P = 0.018) although it was also found that some patients had elevated CTCs without an elevated CEA. No correlation with the time interval between detection of primary tumor and appearance of secondary (metastatic) tumor(s) was found. CTC counts did not correlate with the presence of lung or liver metastases, i.e. a number of mCRC patients with lung or liver metastases had a count of zero CTCs at baseline. We also noted no correlation between CTC number and the status of KRAS or BRAF mutation. CTC counts dropped immediately after the start of chemotherapy in 11 out of 21 patients, and also reduced from the baseline at the end of chemotherapy in 5 out of 10 patients. Six of 7 patients who started with 3 or more CTCs in 7.5 ml at baseline also showed a final CTC reduction at the end of the therapy assessment. Conclusions: Analysis of circulating tumor cells may be of use in monitoring response to therapy in mCRC, either in combination with CEA monitoring or alone when CTCs are elevated but CEA level is not.

Effect of Doxycycline vs Placebo on Retinal Function and Diabetic Retinopathy Progression in Mild to Moderate Nonproliferative Diabetic Retinopathy: A Randomized Proof-of-Concept Clinical Trial

IMPORTANCE Microglia have been associated with inflammatory changes underlying diabetic retinopathy. OBJECTIVE To investigate whether low-dose oral doxycycline monohydrate, a drug capable of inhibiting microglial activation, can improve or slow the deterioration of retinal function and whether it can induce regression or slow progression of diabetic retinopathy in patients with mild to moderate nonproliferative diabetic retinopathy (NPDR). DESIGN, SETTING, AND PARTICIPANTS Randomized, double-masked, 24-month proof-of-concept clinical trial. We randomized 33 patients (from the Penn State Hershey Eye Center) with at least 1 eye with mild to moderate NPDR (Early Treatment Diabetic Retinopathy Study level 20-43) to doxycycline monohydrate, 50 mg/d, or daily placebo for 24 months. MAIN OUTCOMES AND MEASURES Mean change at 24 months compared with baseline in the foveal sensitivity of matrix frequency-doubling perimetry in each treatment group. We also compared the 2 groups with respect to change from baseline to 24 months in functional variables (Humphrey photopic visual field testing using the Swedish interactive thresholding algorithm 24-2 strategy, contrast sensitivity, dark adaptation, visual acuity, and quality of life) and anatomical variables (diabetic retinopathy severity level, area of retinal thickening, central subfield thickness on optical coherence tomography, and macular volume on optical coherence tomography). RESULTS From baseline to month 24, no significant difference was detected between groups with respect to all visual function and anatomical outcomes assessed. CONCLUSIONS AND RELEVANCE Although a link between low-dose oral anti-inflammatory agents and subclinical improvement in inner retinal function has been suggested in patients with severe NPDR or non-high-risk proliferative diabetic retinopathy, the same association was not found in the present study of patients with mild to moderate NPDR. The different findings in the 2 patient populations may relate to a differential effect of doxycycline on different stages of diabetic retinal dysfunction, or the sample size of the present study may be too small to detect a treatment effect of doxycycline in patients with mild to moderate NPDR. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00917553.