Mau-Shin Chi

Apolipoprotein C1 (APOC1) as a novel diagnostic and prognostic biomarker for lung cancer: A marker phase I trial

Tumor cells continuously evolve over time in response to host pressures. However, explanations as to how tumor cells are influenced by the inflammatory tumor microenvironment over time are, to date, poorly defined. We hypothesized that prognostic biomarkers could be obtained by exploring the expression of inflammation‐associated genes between early and late stage lung cancer tumor samples.

Simultaneous activation and inhibition of autophagy sensitizes cancer cells to chemotherapy.

While combined chemotherapy (CT) with an autophagy inducer and an autophagy inhibitor appears paradoxical, it may provide a more effective perturbation of autophagy pathways. We used two dissimilar cell lines to test the hypothesis that autophagy is the common denominator of cell fate after CT. HA22T cells are characterized by CT-induced apoptosis and use autophagy to prevent cell death, while Huh7.5.1 cells exhibit sustained autophagic morphology after CT. Combined CT and rapamycin treatment resulted in a better combination index (CI) in Huh7.5.1 cells than combined CT and chloroquine, while the reverse was true in HA22T cells. The combination of 3 drugs (triplet drug treatment) had the best CI. After triplet drug treatment, HA22T cells switched from protective autophagy to mitochondrial membrane permeabilization and endoplasmic reticulum stress response-induced apoptosis, while Huh7.5.1 cells intensified autophagic lethality. Most importantly, both cell lines showed activation of Akt after CT, while the triplet combination blocked Akt activation through inhibition of phospholipid lipase D activity. This novel finding warrants further investigation as a broad chemosensitization strategy.

Reciprocal Complementation of the Tumoricidal Effects of Radiation and Natural Killer Cells

The tumor microenvironment is a key determinant for radio-responsiveness. Immune cells play an important role in shaping tumor microenvironments; however, there is limited understanding of how natural killer (NK) cells can enhance radiation effects. This study aimed to assess the mechanism of reciprocal complementation of radiation and NK cells on tumor killing. Various tumor cell lines were co-cultured with human primary NK cells or NK cell line (NK-92) for short periods and then exposed to irradiation. Cell proliferation, apoptosis and transwell assays were performed to assess apoptotic efficacy and cell viability. Western blot analysis and immunoprecipitation methods were used to determine XIAP (X-linked inhibitor of apoptosis protein) and Smac (second mitochondria-derived activator of caspase) expression and interaction in tumor cells. Co-culture did not induce apoptosis in tumor cells, but a time- and dose-dependent enhancing effect was found when co-cultured cells were irradiated. A key role for caspase activation via perforin/granzyme B (Grz B) after cell-cell contact was determined, as the primary radiation enhancing effect. The efficacy of NK cell killing was attenuated by upregulation of XIAP to bind caspase-3 in tumor cells to escape apoptosis. Knockdown of XIAP effectively potentiated NK cell-mediated apoptosis. Radiation induced Smac released from mitochondria and neutralized XIAP and therefore increased the NK killing. Our findings suggest NK cells in tumor microenvironment have direct radiosensitization effect through Grz B injection while radiation enhances NK cytotoxicity through triggering Smac release.

Double autophagy modulators reduce 2-deoxyglucose uptake in sarcoma patients

Rationale According to the metabolic symbiosis model, cancer stromal fibroblasts could be hijacked by surrounding cancer cells into a state of autophagy with aerobic glycolysis to help provide recycled nutrients. The purpose of this study was to investigate whether combined treatment with the autophagy inhibitor: hydroxychloroquine (HCQ) and the autophagy inducer: sirolimus (rapamycin, Rapa) would reduce glucose utilization in sarcoma patients. Methods Ten sarcoma patients who failed first-line treatment were enrolled in this study. They were treated with 1 mg of Rapa and 200 mg of HCQ twice daily for two weeks. The standardized uptake values (SUV) from pretreatment and posttreatment [18F]-fluorodeoxyglucose positron emission tomography (FDG PET) scans were reviewed, and changes from the baseline SUVmax were evaluated. Results Based on FDG PET response criteria, six patients had a partial response; three had stable disease, and one had progressive disease. Nevertheless, none of them showed a reduction in tumor volume. The mean SUVmax reduction in the 34 lesions evaluated was − 19.6% (95% CI = −30.1% to −9.1%), while the mean volume change was +16.4% (95% CI = +5.8% to + 27%). Only grade 1 toxicities were observed. Elevated serum levels of lactate dehydrogenase were detected after treatment in most metabolic responders. Conclusions The results of reduced SUVmax without tumor volume reduction after two weeks of Rapa and HCQ treatment may indicate that non-proliferative glycolysis occurred mainly in the cancer associated fibroblast compartment, and decreased glycolytic activity was evident from Rapa + HCQ double autophagy modulator treatment.

Serum amyloid a as a predictive marker for radiation pneumonitis in lung cancer patients.

PURPOSE To investigate serum markers associated with radiation pneumonitis (RP) grade ≥3 in patients with lung cancer who were treated with radiation therapy. METHODS AND MATERIALS Pretreatment serum samples from patients with stage Ib-IV lung cancer who developed RP within 1 year after radiation therapy were analyzed to identify a proteome marker able to stratify patients prone to develop severe RP by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Dosimetric parameters and 3 biological factors were compared. RESULTS Serum samples from 16 patients (28%) with severe RP (grade 3-4) and 42 patients (72%) with no or mild RP (grade 0-2) were collected for analysis. All patients received a median of 54 Gy (range, 42-70 Gy) of three-dimensional conformal radiation therapy with a mean lung dose (MLD) of 1502 cGy (range, 700-2794 cGy). An m/z peak of 11,480 Da was identified by SELDI-TOF-MS, and serum amyloid A (SAA) was the primary splitter serum marker. The receiver operating characteristic area under the curve of SAA (0.94; 95% confidence interval [CI], 0.87-1.00) was higher than those of C-reactive protein (0.83; 95% CI, 0.72-0.94), interleukin-6 (0.79; 95% CI, 0.65-0.94), and MLD (0.57; 95% CI, 0.37-0.77). The best sensitivity and specificity of combined SAA and MLD for predicting RP were 88.9% and 96.0%, respectively. CONCLUSIONS Baseline SAA could be used as an auxiliary marker for predicting severe RP. Extreme care should be taken to limit the lung irradiation dose in patients with high SAA.

Temozolomide, sirolimus and chloroquine is a new therapeutic combination that synergizes to disrupt lysosomal function and cholesterol homeostasis in GBM cells

Glioblastoma (GBM) cells are characterized by high phagocytosis, lipogenesis, exocytosis activities, low autophagy capacity and high lysosomal demand are necessary for survival and invasion. The lysosome stands at the cross roads of lipid biosynthesis, transporting, sorting between exogenous and endogenous cholesterol. We hypothesized that three already approved drugs, the autophagy inducer, sirolimus (rapamycin, Rapa), the autophagy inhibitor, chloroquine (CQ), and DNA alkylating chemotherapy, temozolomide (TMZ) could synergize against GBM. This repurposed triple therapy combination induced GBM apoptosis in vitro and inhibited GBM xenograft growth in vivo. Cytotoxicity is caused by induction of lysosomal membrane permeabilization and release of hydrolases, and may be rescued by cholesterol supplementation. Triple treatment inhibits lysosomal function, prevents cholesterol extraction from low density lipoprotein (LDL), and causes clumping of lysosome associated membrane protein-1 (LAMP-1) and lipid droplets (LD) accumulation. Co-treatment of the cell lines with inhibitor of caspases and cathepsin B only partially reverse of cytotoxicities, while N-acetyl cysteine (NAC) can be more effective. A combination of reactive oxygen species (ROS) generation from cholesterol depletion are the early event of underling mechanism. Cholesterol repletion abolished the ROS production and reversed the cytotoxicity from QRT treatment. The shortage of free cholesterol destabilizes lysosomal membranes converting aborted autophagy to apoptosis through either direct mitochondria damage or cathepsin B release. This promising anti-GBM triple therapy combination severely decreases mitochondrial function, induces lysosome-dependent apoptotic cell death, and is now poised for further clinical testing and validation.

Recombinant heat shock protein 70 in combination with radiotherapy as a source of tumor antigens to improve dendritic cell immunotherapy.

Local radiotherapy (RT) plus intratumoral dendritic cell (DC) injection can mediate immunological response. We hypothesized that co-injection of exogenous recombinant heat shock protein 70 (rHsp70) in combination with RT-DC could be as effective as co-injection of HSP-peptide for evoking specific immune response. rHsp70-prostate-specific antigen (rHSP70C′-PSA) and α-fetoprotein (rHSP70C′-AFP) were used to compare specific response. Growth inhibition of the tumor and the systemic anti-tumor immune response were measured on CT26/PSA and CT26/AFP mice model. Intratumoral co-injection of rHsp70 and DC into the irradiated tumor site induced a more potent anti-tumor immune response than injection of DC alone. rHsp70 was as effective as rHsp70C′-PSA or rHsp70C′-AFP in inducing a tumor-specific cytotoxic T lymphocyte response or tumor growth delay. These results demonstrate that co-administration with rHsp70 and RT could be a simple and effective source of tumor antigens to achieve RT-DC immunotherapy protocol and easy to apply in clinical use.

Abstract 5611: A synthetic lethality combination with chloroquine and rapamycin on chemosensitization

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Chemoresistance is the main obstacle of successful cancer therapy. Autophagy may either enhance cancer cell death or activate chemoresistance. Simultaneous pushing and blocking of autophagy may be a valid strategy for overcoming drug resistance and improving responses. We hypothesized that simultaneous forcing hepatoma cells synchronized in a pro-autophagic status by rapamycin while blocking the final autophagic pathway by chloroquine is an example of synthetic lethality in synergism. Co-administration of the chemotherapeutic agents with rapamycin and chloroquine in 3 hepatoma cell lines have all demonstrated synergistic effects including 5-fluorouracil, navelbine, taxotere or cisplatin. Increasing doses of rapamycin and chloroquine resulted in a significant decrease of ED50 of different chemodrugs. Althrough hepatoma cell lines expressed difference status of autophage during various chemodrug treatments, co-administration of rapamycin and chloroquine could synchronize them on the pro-autophagic status while preveting them ensuring to the final autophagic status in all cell line tested. The combination of rapamycin and chloroquine and chemotherapeutic agents resulted in increased apoptotic cell death compared to chemotherapeutic agents alone. All of the observed synergistic chemosensitization effect from combination of chloroquine and rapamycin is more obvious than either agent alone. In vivo experiments are underway. If these synergistic effects can be reproduced in vivo, the combination of rapamycin and chloroquine may provide a valid reversing resistance strategy in treatment refractory cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5611. doi:1538-7445.AM2012-5611

In vitro comparison of conventional hyperthermia and modulated electro-hyperthermia.

Radiofrequency-induced hyperthermia (HT) treatments for cancer include conventional capacitive coupling hyperthermia (cCHT) and modulated electro-hyperthermia (mEHT). In this study, we directly compared these methods with regard to in vitro cytotoxicity and mechanisms of action under isothermal conditions. Hepatoma (HepG2) cells were exposed to HT treatment (42°C for 30 min) using mEHT, cCHT or a water bath. mEHT produced a much higher apoptosis rate (43.1% ± 5.8%) than cCHT (10.0% ± 0.6%), the water bath (8.4% ± 1.7%) or a 37°C control (6.6% ± 1.1%). The apoptosis-inducing effect of mEHT at 42°C was similar to that achieved with a water bath at 46°C. mEHT also increased expression of caspase-3, 8 and 9. All three hyperthermia methods increased intracellular heat shock protein 70 (Hsp70) levels, but only mEHT greatly increased the release of Hsp70 from cells. Calreticulin and E-cadherin levels in the cell membrane also increased after mEHT treatment, but not after cCHT or water bath. These results suggest that mEHT selectively deposits energy on the cell membrane and may be a useful treatment modality that targets cancer cell membranes.

REPEAT IRRADIATION OF RECURRENT HEPATOCELLULAR CARCINOMA - A SINGLE INSTITUTE EXPERIENCE

Purpose: Radiotherapy is recognized as an effective local treatment for hepatocellular carcinoma, but limited evidence exists for its efficacy in repeat liver irradiation. When other treatment modalities have been exhausted, repeat irradiation may be the only option. A single institution experience of repeat irradiation of hepatocellular carcinoma is reported here. Materials and Methods: Between 2003 and 2013, 19 patients (M/F = 17/2; median age = 64.4 years) with recurrent hepatocellular carcinoma (8 in-field and 11 out-field) and having had previous radiotherapy, were treated with repeat irradiation. The primary eligibility criteria for selecting these patients for repeat liver irradiation was normal liver volume greater than 700 mL. The median equivalent dose in 2 Gy (EQD2) delivered to tumors was 47.5 Gy (range: 30-71 Gy). A proportion of patients had concurrent and maintenance angiogenic blockades. Univariate analyses were used to identify statistically significant prognostic factors. Results: Among the 19 patients, complete response was achieved in 4 patients, and partial response in 6. The median overall survival for the entire series was 10.3 months. Overall survival rates at 1 and 2 years were 47% and 26% respectively. The presence of either longer treatment intervals between radiotherapies (＞ 6.2 months) or use of angiogenic blockades was associated with longer overall survival on univariate analysis. The incidence of radiation induced liver injury was 11%. Conclusions: Repeat hepatic irradiation is possible where the necessary dosimetric parameters can be met. Patients with longer intervals between radiotherapies may achieve longer survival rates in comparison to those without. Survival benefit is attainable with concomitant and maintenance angiogenic blockades.