Yasushi Ino

Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics.

ABSTRACT Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16INK4a cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16INK4a-negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT+ keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16INK4a expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16INK4a-dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems.

Autocrine TGF-β Signaling Maintains Tumorigenicity of Glioma-Initiating Cells through Sry-Related HMG-Box Factors

Despite aggressive surgery, radiotherapy, and chemotherapy, treatment of malignant glioma remains formidable. Although the concept of cancer stem cells reveals a new framework of cancer therapeutic strategies against malignant glioma, it remains unclear how glioma stem cells could be eradicated. Here, we demonstrate that autocrine TGF-beta signaling plays an essential role in retention of stemness of glioma-initiating cells (GICs) and describe the underlying mechanism for it. TGF-beta induced [corrected] expression of Sox2, a stemness gene, and this induction was mediated by Sox4, a direct TGF-beta target gene. Inhibitors of TGF-beta signaling drastically deprived tumorigenicity of GICs by promoting their differentiation, and these effects were attenuated in GICs transduced with Sox2 or Sox4. Furthermore, GICs pretreated with TGF-beta signaling inhibitor exhibited less lethal potency in intracranial transplantation assay. These results identify an essential pathway for GICs, the TGF-beta-Sox4-Sox2 pathway, whose disruption would be a therapeutic strategy against gliomas.

Glioma-initiating Cells Retain Their Tumorigenicity through Integration of the Sox Axis and Oct4 Protein

Background: Glioma-initiating cells are underlying causes of development and progression of glioblastoma. Results: Depletion of Oct4 expression suppresses tumorigenic activity of glioma-initiating cells through down-regulation of Sox2. Conclusion: Oct4 maintains tumorigenicity of glioma-initiating cells in cooperation with the Sox axis. Significance: This study uncovers the transcriptional network of stemness genes in cancer-initiating cells. Although the concept of cancer stem cells or cancer-initiating cells had created a new paradigm for the treatment of malignant tumors, it remains unclear how cancer-initiating cells can be eradicated. We have previously reported that the transforming growth factor-β (TGF-β)-Sox4-Sox2 pathway is essential for glioma-initiating cells to retain their stemness, and inhibition of TGF-β signaling may lead to differentiation of glioma-initiating cells (Ikushima, H., Todo, T., Ino, Y., Takahashi, M., Miyazawa, K., and Miyazono, K. (2009) Cell Stem Cell 5, 504–514). Here we demonstrate that Oct4 plays essential roles in retention of the stemness properties of glioma-initiating cells through positive regulation of Sox2 expression. We also show that, in glioma-initiating cells, Oct4 is associated with Sox4 and that Oct4-Sox4 complexes cooperatively activate the enhancer activity of the SOX2 gene. In contrast, in fetal neural progenitor cells, Sox2 expression is enhanced by transcriptional complex containing Sox2 protein itself, and this self-reinforcing loop of Sox2 appears to be disrupted in glioma-initiating cells, suggesting that Sox2 expression in glioma-initiating cells is differently regulated from that in neural progenitor cells. Our findings reveal differences between glioma-initiating cells and fetal neural progenitor cells and may open the way to depriving glioma-initiating cells of tumorigenic activity without affecting normal tissues.

Oncolytic virus therapy: A new era of cancer treatment at dawn

Oncolytic virus therapy is perhaps the next major breakthrough in cancer treatment following the success in immunotherapy using immune checkpoint inhibitors. Oncolytic viruses are defined as genetically engineered or naturally occurring viruses that selectively replicate in and kill cancer cells without harming the normal tissues. T‐Vec (talimogene laherparepvec), a second‐generation oncolytic herpes simplex virus type 1 (HSV‐1) armed with GM‐CSF, was recently approved as the first oncolytic virus drug in the USA and Europe. The phase III trial proved that local intralesional injections with T‐Vec in advanced malignant melanoma patients can not only suppress the growth of injected tumors but also act systemically and prolong overall survival. Other oncolytic viruses that are closing in on drug approval in North America and Europe include vaccinia virus JX‐594 (pexastimogene devacirepvec) for hepatocellular carcinoma, GM‐CSF‐expressing adenovirus CG0070 for bladder cancer, and Reolysin (pelareorep), a wild‐type variant of reovirus, for head and neck cancer. In Japan, a phase II clinical trial of G47∆, a third‐generation oncolytic HSV‐1, is ongoing in glioblastoma patients. G47∆ was recently designated as a “Sakigake” breakthrough therapy drug in Japan. This new system by the Japanese government should provide G47∆ with priority reviews and a fast‐track drug approval by the regulatory authorities. Whereas numerous oncolytic viruses have been subjected to clinical trials, the common feature that is expected to play a major role in prolonging the survival of cancer patients is an induction of specific antitumor immunity in the course of tumor‐specific viral replication. It appears that it will not be long before oncolytic virus therapy becomes a standard therapeutic option for all cancer patients.

Triple combination of oncolytic herpes simplex virus-1 vectors armed with interleukin-12, interleukin-18, or soluble B7-1 results in enhanced antitumor efficacy.

Conditionally replicating herpes simplex virus-1 (HSV-1) vectors are promising therapeutic agents for cancer. Insertion of therapeutic transgenes into the viral genome should confer desired anticancer functions in addition to oncolytic activities. Herein, using bacterial artificial chromosome and two recombinase-mediated recombinations, we simultaneously created four “armed” oncolytic HSV-1, designated vHsv-B7.1-Ig, vHsv-interleukin (IL)-12, vHsv-IL-18, and vHsv-null, which express murine soluble B7.1 (B7.1-Ig), murine IL-12, murine IL-18, and no transgene, respectively. These vHsv vectors possess deletions in the γ34.5 genes and contain the green fluorescent protein gene as a histochemical marker and the immunostimulatory transgene inserted in the deleted ICP6 locus. The vHsv showed similar replicative capabilities in vitro. The in vivo efficacy was tested in A/J mice harboring s.c. tumors of syngeneic and poorly immunogenic Neuro2a neuroblastoma. The triple combination of vHsv-B7.1-Ig, vHsv-IL-12, and vHsv-IL-18 exhibited the highest efficacy among all single vHsv or combinations of two viruses. Combining 1 × 105 plaque-forming units each of the three armed viruses showed stronger antitumor activities than any single armed virus at 3 × 105 plaque-forming units in inoculated tumors as well as in noninoculated remote tumors. Studies using athymic mice indicated that this enhancement of antitumor efficacy was likely mediated by T-cell immune responses. The combined use of multiple oncolytic HSV-1 armed with different immunostimulatory genes may be a useful strategy for cancer therapy.

High-dose conformal radiotherapy for supratentorial malignant glioma: a historical comparison

BACKGROUND Although radiotherapy remains the main postoperative treatment for patients with malignant glioma, modifications to regimens have not improved the poor outlook of patients with this disease. We aimed to investigate whether high-dose conformal radiotherapy improves the survival of patients with supratentorial malignant glioma compared with conventional radiotherapy. METHODS 29 patients with anaplastic astrocytoma and 61 patients with glioblastoma who received high-dose conformal radiotherapy during 1990-2002 were compared with 34 patients with anaplastic astrocytoma and 60 patients with glioblastoma who received conventional 60 Gy radiotherapy during 1979-89. 77 of the 90 patients receiving high-dose radiotherapy were given 80 Gy; the remaining 13 patients, all with glioblastoma, received 90 Gy. Radiotherapy was planned on the basis of images taken before surgery, and doses were delivered in 2 Gy per fraction per day for 5 days a week. Hazard ratios for death were calculated with a Cox model, and were adjusted for age, Karnofsky performance scale, tumour size, and extent of resection. FINDINGS Patients who received high-dose radiotherapy had significantly longer overall survival compared with those who received conventional radiotherapy (adjusted hazard ratio 0.30 [95% CI 0.12-0.76], p=0.011 for anaplastic astrocytoma and 0.49 [0.28-0.87], p=0.014 for glioblastoma). Patients with anaplastic astrocytoma in the high-dose group have not yet reached median survival; median survival in the conventional radiotherapy group was 22.3 months (95% CI 20.6-24.0). 5-year survival was 51.3% (29.2-73.4) for the high-dose group and 14.7% (0.0-30.0) for the conventional group. Median survival in patients with glioblastoma was 16.2 months (12.8-19.6) for the high-dose group and 12.4 months (10.0-14.8) for the conventional group. 2-year survival was 38.4% (23.5-53.3) for the high-dose group and 11.4% (0.0-25.3) for the conventional group. Survival did not differ between those that received 80 Gy radiotherapy and those that received 90 Gy (hazard ratio 0.94 [95% CI 0.42-2.12]). The higher frequency of radiation-induced white matter abnormality in the high-dose group compared with the conventional radiotherapy group did not lead to increased disability. INTERPRETATION High-dose, standard-fractionated radiotherapy shows potential as the main postoperative treatment for patients with supratentorial malignant glioma.

5-Hydroxymethylcytosine Plays a Critical Role in Glioblastomagenesis by Recruiting the CHTOP-Methylosome Complex

The development of cancer is driven not only by genetic mutations but also by epigenetic alterations. Here, we show that TET1-mediated production of 5-hydroxymethylcytosine (5hmC) is required for the tumorigenicity of glioblastoma cells. Furthermore, we demonstrate that chromatin target of PRMT1 (CHTOP) binds to 5hmC. We found that CHTOP is associated with an arginine methyltransferase complex, termed the methylosome, and that this promotes the PRMT1-mediated methylation of arginine 3 of histone H4 (H4R3) in genes involved in glioblastomagenesis, including EGFR, AKT3, CDK6, CCND2, and BRAF. Moreover, we found that CHTOP and PRMT1 are essential for the expression of these genes and that CHTOP is required for the tumorigenicity of glioblastoma cells. These results suggest that 5hmC plays a critical role in glioblastomagenesis by recruiting the CHTOP-methylosome complex to selective sites on the chromosome, where it methylates H4R3 and activates the transcription of cancer-related genes.

Extended field stereotactic radiosurgery for recurrent glioblastoma.

Stereotactic radiosurgery (SRS) is among the few therapeutic options for glioblastoma that recurs after standard radiation and chemotherapy, but its efficacy has been limited.

Long-term control of disseminated pleomorphic xanthoastrocytoma with anaplastic features by means of stereotactic irradiation

Pleomorphic xanthoastrocytoma (PXA) is a rare astrocytic neoplasm of the brain. Some PXAs are accompanied by anaplastic features and are difficult to manage because of frequent recurrences that lead to early death. No previous reports have demonstrated consistent efficacy of adjuvant radiotherapy or chemotherapy for this disease. We report a case of PXA with anaplastic features treated with stereotactic irradiation (STI) that resulted in long-term control of repeatedly recurring nodules throughout the neuraxis. A 47-year-old woman presented with an epileptic seizure due to a large tumor in the right frontal lobe. The tumor was resected and diagnosed as PXA with anaplastic features. Sixteen months later, a relapse at the primary site was noted and treated with stereotactic radiosurgery using Gamma Knife. Two years later, the patient developed a tumor nodule in the cervical spinal cord that histologically corresponded to a small-cell glioma with high cellularity and prominent MIB-1 (mindbomb homolog 1) labeling. In the following months, multiple nodular lesions appeared throughout the CNS, and STI was performed six times for eight intracranial lesions using Gamma Knife and twice using a linear accelerator, for three spinal cord lesions in total. All lesions treated with STI were well controlled, and the patient was free from symptomatic progression for 50 months. However, diffuse dissemination along the craniospinal axis eventually progressed, and she died 66 months after initial diagnosis. Autopsy showed that the nodules remained well demarcated from the surrounding nervous system tissue. STI may be an effective therapeutic tool for controlling nodular dissemination of PXA with anaplastic features.

The critical role of cyclin D2 in cell cycle progression and tumorigenicity of glioblastoma stem cells.

Cancer stem cells are believed to be responsible for tumor initiation and development. Much current research on human brain tumors is focused on the stem-like properties of glioblastoma stem cells (GSCs). However, little is known about the molecular mechanisms of cell cycle regulation that discriminate between GSCs and differentiated glioblastoma cells. Here we show that cyclin D2 is the cyclin that is predominantly expressed in GSCs and suppression of its expression by RNA interference causes G1 arrest in vitro and growth retardation of GSCs xenografted into immunocompromised mice in vivo. We also demonstrate that the expression of cyclin D2 is suppressed upon serum-induced differentiation similar to what was observed for the cancer stem cell marker CD133. Taken together, our results demonstrate that cyclin D2 has a critical role in cell cycle progression and the tumorigenicity of GSCs.