mTOR Inhibitors Synergize on Regression, Reversal of Gene Expression, and Autophagy in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) affects more than half a million people worldwide and is the third most common cause of cancer deaths. Because mammalian target of rapamycin (mTOR) signaling is up-regulated in 50% of HCCs, we compared the effects of the U.S. Food and Drug Administration-approved mTOR-allosteric inhibitor, RAD001, with a new-generation phosphatidylinositol 3-kinase/mTOR adenosine triphosphate-site competitive inhibitor, BEZ235. Unexpectedly, the two drugs acted synergistically in inhibiting the proliferation of cultured HCC cells. The synergistic effect closely paralleled eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) dephosphorylation, which is implicated in the suppression of tumor cell proliferation. In a mouse model approximating human HCC, the drugs in combination, but not singly, induced a marked regression in tumor burden. However, in the tumor, BEZ235 alone was as effective as the combination in inhibiting 4E-BP1 phosphorylation, which suggests that additional target(s) may also be involved. Microarray analyses revealed a large number of genes that reverted to normal liver tissue expression in mice treated with both drugs, but not either drug alone. These analyses also revealed the down-regulation of autophagy genes in tumors compared to normal liver. Moreover, in HCC patients, altered expression of autophagy genes was associated with poor prognosis. Consistent with these findings, the drug combination had a profound effect on UNC51-like kinase 1 (ULK1) dephosphorylation and autophagy in culture, independent of 4E-BP1, and in parallel induced tumor mitophagy, a tumor suppressor process in liver. These observations have led to an investigator-initiated phase 1B-2 dose escalation trial with RAD001 combined with BEZ235 in patients with HCC and other advance

Single-Cell Phosphoproteomics Resolves Adaptive Signaling Dynamics and Informs Targeted Combination Therapy in Glioblastoma

Intratumoral heterogeneity of signaling networks may contribute to targeted cancer therapy resistance, including in the highly lethal brain cancer glioblastoma (GBM). We performed single-cell phosphoproteomics on a patient-derived in vivo GBM model of mTOR kinase inhibitor resistance and coupled it to an analytical approach for detecting changes in signaling coordination. Alterations in the protein signaling coordination were resolved as early as 2.5 days after treatment, anticipating drug resistance long before it was clinically manifest. Combination therapies were identified that resulted in complete and sustained tumor suppression in vivo. This approach may identify actionable alterations in signal coordination that underlie adaptive resistance, which can be suppressed through combination drug therapy, including non-obvious drug combinations

Discrimination between low-grade oligodendrogliomas and diffuse astrocytoma with the aid of 11C-methionine positron emission tomography.

OBJECT: The diagnostic usefulness of (11)C-methionine PET scans in gliomas is still controversial. The authors investigated the clinical significance of (11)C-methionine PET findings in preoperative diagnosis of histological type and grade.\nMETHODS: The tissue uptake of (11)C-methionine was assessed using PET in 70 patients with histologically confirmed intracerebral gliomas. The ratio of maximum standard uptake values in tumor areas to the mean standard uptake values in the contralateral normal brain tissue (tumor/normal tissue [T/N] ratio) was calculated and correlated with tumor type, histological grade, contrast enhancement on MR imaging, Ki 67 labeling index, and 1p/19q status.\nRESULTS: The T/N ratio was significantly increased as tumor grade advanced in astrocytic tumors (WHO Grade II vs Grade III, p = 0.0011; Grade III vs Grade IV, p = 0.0007). Among Grade II gliomas, the mean T/N ratio was significantly higher in oligodendroglial tumors than in diffuse astrocytomas (DAs) (p < 0.0001). All T/N ratios for oligodendroglial tumors were &#8805; 1.46, and those for DA were consistently < 1.46, with the exception of 2 cases of gemistocytic astrocytoma. The Ki 67 labeling index significantly correlated with T/N ratio in astrocytic tumors, but not in oligodendrogliomas. Oligodendroglial tumors without 1p/19q deletion had a significantly higher T/N ratio than those with the codeletion. In combination with Gd-enhanced MR imaging, 67% of nonenhanced tumors with a T/N ratio of &#8805; 1.46 were proved to be Grade II oligodendrogliomas.\nCONCLUSIONS: These results clearly show that (11)C-methionine PET T/N ratios in Grade II oligodendrogliomas were higher than those in DAs independently of their proliferative activity. This information contributes to preoperative differential diagnoses of histological type, especially in suspected low-grade gliomas

Circulating anti-filamin C autoantibody as a potential serum biomarker for low-grade gliomas

BACKGROUND: Glioma is the most common primary malignant central nervous system tumor in adult, and is usually not curable due to its invasive nature. Establishment of serum biomarkers for glioma would be beneficial both for early diagnosis and adequate therapeutic intervention. Filamins are an actin cross-linker and filamin C (FLNC), normally restricted in muscle tissues, offers many signaling molecules an essential communication fields. Recently, filamins have been considered important for tumorigenesis in cancers. METHODS: We searched for novel glioma-associated antigens by serological identification of antigens utilizing recombinant cDNA expression cloning (SEREX), and found FLNC as a candidate protein. Tissue expressions of FLNC (both in normal and tumor tissues) were examined by immunohistochemistry and quantitative RT-PCR analyses. Serum anti-FLNC autoantibody level was measured by ELISA in normal volunteers and in the patients with various grade gliomas. RESULTS: FLNC was expressed in glioma tissues and its level got higher as tumor grade advanced. Anti-FLNC autoantibody was also detected in the serum of glioma patients, but its levels were inversely correlated with the tissue expression. Serum anti-FLNC autoantibody level was significantly higher in low-grade glioma patients than in high-grade glioma patients or in normal volunteers, which was confirmed in an independent validation set of patients’ sera. The autoantibody levels in the patients with meningioma or cerebral infarction were at the same level of normal volunteers, and they were significantly lower than that of low-grade gliomas. Total IgG and anti-glutatione S-transferase (GST) antibody level were not altered among the patient groups, which suggest that the autoantibody response was specific for FLNC. CONCLUSIONS: The present results suggest that serum anti-FLNC autoantibody can be a potential serum biomarker for early diagnosis of low-grade gliomas while it needs a large-scale clinical study

Dual Inhibition of mTOR in hepatocellular carcinoma induces tumor regression, return to normal liver gene expression and autophagy

Hepatocellular carcinoma (HCC) affects more than half a million people worldwide and is the third most common cause of cancer deaths. Given the need for novel therapies and that mTOR signaling is upregulated in 50% of HCCs, we compared the effects of the FDA-approved mTOR-allosteric inhibitor, RAD001, with a new generation PI3K/mTOR ATP-site competitive inhibitor, BEZ235. Unexpectedly, the two drugs acted synergistically in inhibiting proliferation of cultured HCC cells, which closely paralleled 4E-BP1 dephosphorylation. In a mouse model approximating human HCC with poor prognosis, the two drugs in combination, but not as single agents, induced a dramatic regression in tumor development. However, BEZ235 was almost as effective as the combination in inhibiting 4E-BP1 phosphorylation. Importantly, microarray analyses revealed a large and unique number of genes reverting to normal liver tissue expression levels in tumors treated with both drugs, but not with either drug alone. These analyses also revealed the down regulation of a number of autophagy genes in tumors compared to normal liver. This observation led to the finding that the drug combination has a profound effect on ULK1 phosphorylation and autophagy in culture and in parallel induces mitophagy in tumors, a process which acts as a tumor suppressor in liver. As drug synergy is achieved at low doses of both drugs, this may also decrease potential toxicity, while increasing target specificity. These observations have led to an investigator initiated Phase 1B-2 dose escalation trial with RAD001 combined with BEZ235 in patients with advanced solid tumors, including HCC

mTOR Complex 2 Controls Glycolytic Metabolism in Glioblastoma through FoxO Acetylation and Upregulation of c-Myc

Aerobic glycolysis (the Warburg effect) is a core hallmark of cancer, but the molecular mechanisms underlying it remain unclear. Here, we identify an unexpected central role for mTORC2 in cancer metabolic reprogramming where it controls glycolytic metabolism by ultimately regulating the cellular level of c-Myc. We show that mTORC2 promotes inactivating phosphorylation of class IIa histone deacetylases, which leads to the acetylation of FoxO1 and FoxO3, and this in turn releases c-Myc from a suppressive miR-34c-dependent network. These central features of activated mTORC2 signaling, acetylated FoxO, and c-Myc levels are highly intercorrelated in clinical samples and with shorter survival of GBM patients. These results identify a specific, Akt-independent role for mTORC2 in regulating glycolytic metabolism in cancer

Serum anti-PCK1 antibody levels are a prognostic factor for patients with diabetes mellitus

Abstract Background Autoantibodies develop in autoimmune diseases, cancer, diabetes mellitus (DM), and atherosclerosis-related diseases. However, autoantibody biomarkers have not been successfully examined for diagnosis and therapy. Methods Serological identification of antigens through recombinant cDNA expression cloning (SEREX) was used for primary screening of antigens. The cDNA product was expressed in bacteria and purified. Amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) was used to evaluate antibody levels in serum samples. Results Phosphoenolpyruvate carboxykinase 1 (PCK1) was recognized as an antigen by serum IgG antibodies in the sera of patients with atherosclerosis. AlphaLISA showed significantly higher serum antibody levels against recombinant PCK1 protein in patients with DM and cardiovascular disease than in healthy donors, but not in those with acute ischemic stroke, transient ischemic attack, or obstructive sleep apnea syndrome. The area under the receiver operating characteristic curve for anti-PCK1 antibodies was 0.7024 for DM. The serum anti-PCK1 antibody levels were associated with age, platelet count, and blood pressure. Anti-PCK1-antibody-positive patients showed significantly lower overall survival than the negative patients. Conclusions Serum anti-PCK1 antibody levels were found to be associated with DM. The anti-PCK1 antibody marker is useful for predicting the overall survival of patients with DM

mTOR Inhibitors Synergize on Regression, Reversal of Gene Expression, and Autophagy in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) affects more than half a million people worldwide and is the third most common cause of cancer deaths. Because mammalian target of rapamycin (mTOR) signaling is up-regulated in 50% of HCCs, we compared the effects of the U.S. Food and Drug Administration-approved mTOR-allosteric inhibitor, RAD001, with a new-generation phosphatidylinositol 3-kinase/mTOR adenosine triphosphate-site competitive inhibitor, BEZ235. Unexpectedly, the two drugs acted synergistically in inhibiting the proliferation of cultured HCC cells. The synergistic effect closely paralleled eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) dephosphorylation, which is implicated in the suppression of tumor cell proliferation. In a mouse model approximating human HCC, the drugs in combination, but not singly, induced a marked regression in tumor burden. However, in the tumor, BEZ235 alone was as effective as the combination in inhibiting 4E-BP1 phosphorylation, which suggests that additional target(s) may also be involved. Microarray analyses revealed a large number of genes that reverted to normal liver tissue expression in mice treated with both drugs, but not either drug alone. These analyses also revealed the down-regulation of autophagy genes in tumors compared to normal liver. Moreover, in HCC patients, altered expression of autophagy genes was associated with poor prognosis. Consistent with these findings, the drug combination had a profound effect on UNC51-like kinase 1 (ULK1) dephosphorylation and autophagy in culture, independent of 4E-BP1, and in parallel induced tumor mitophagy, a tumor suppressor process in liver. These observations have led to an investigator-initiated phase 1B-2 dose escalation trial with RAD001 combined with BEZ235 in patients with HCC and other advance