Shuhei Suzuki

JNK contributes to temozolomide resistance of stem-like glioblastoma cells via regulation of MGMT expression

While elimination of the cancer stem cell population is increasingly recognized as a key to successful treatment of cancer, the high resistance of cancer stem cells to conventional chemoradiotherapy remains a therapeutic challenge. O6-methylguanine DNA methyltransferase (MGMT), which is frequently expressed in cancer stem cells of glioblastoma, has been implicated in their resistance to temozolomide, the first-line chemotherapeutic agent against newly diagnosed glioblastoma. However, much remains unknown about the molecular regulation that underlies MGMT expression and temozolomide resistance of glioblastoma cancer stem cells. Here, we identified JNK as a novel player in the control of MGMT expression and temozolomide resistance of glioblastoma cancer stem cells. We showed that inhibition of JNK, either pharmacologically or by RNA interference, in stem-like glioblastoma cells derived directly from glioblastoma tissues reduces their MGMT expression and temozolomide resistance. Importantly, sensitization of stem-like glioblastoma cells to temozolomide by JNK inhibition was dependent on MGMT expression, implying that JNK controls temozolomide resistance of stem-like glioblastoma cells through MGMT expression. Our findings suggest that concurrent use of JNK inhibitors with temozolomide may be a rational therapeutic approach to effectively target the cancer stem cell population in the treatment of glioblastoma.

Specific role of JNK in the maintenance of the tumor-initiating capacity of A549 human non-small cell lung cancer cells

Deregulation of c-Jun NH2-terminal kinase (JNK) signaling is now increasingly reported in a variety of human malignancies. Non-small cell lung cancer (NSCLC) is among such human malignancies with aberrant JNK activation; yet the exact role(s) of JNK deregulation in NSCLC biology, in particular in vivo, remains unclear. Here, we demonstrated a specific role of JNK in the control of the tumor-initiating capacity of A549 cells derived from human lung adenocarcinoma, a major subtype of NSCLC. Despite its potent inhibitory activity on A549 cell growth in vitro, SP600125, a reversible JNK inhibitor, failed to inhibit the growth of pre-established A549 xenografts in vivo when systemically administered. Nevertheless, the same SP600125 treatment caused a marked reduction in the tumor-initiating population within the A549 tumors, suggesting that JNK may be specifically required in vivo for the maintenance of the tumor-initiating population of tumor cells rather than for proliferation and survival of the entire cell population. Furthermore, A549 cells either pre-treated with SP600125 or transiently transfected with siRNAs against the JNK genes in vitro showed substantially reduced ability to initiate tumor formation upon implantation into nude mice, implying that the cell intrinsic JNK activity of A549 cells is essential for the maintenance of their tumor-initiating capacity. To our knowledge, this is the first demonstration that JNK is involved in the control of the tumor-initiating capacity of NSCLC cells. Our findings also give rise to an intriguing possibility that therapies targeting JNK could contribute to prevention of relapse and/or metastasis of NSCLC through elimination of tumor-initiating cells.

The novel JNK inhibitor AS602801 inhibits cancer stem cells in vitro and in vivo.

A phase 2 clinical trial investigating the efficacy and safety of AS602801, a newly developed JNK inhibitor, in the treatment of inflammatory endometriosis is complete. We are now examining whether AS602801 acts against human cancer cells in vitro and in vivo. In vitro, AS602801 exhibited cytotoxicity against both serum-cultured non-stem cancer cells and cancer stem cells derived from human pancreatic cancer, non-small cell lung cancer, ovarian cancer and glioblastoma at concentrations that did not decrease the viability of normal human fibroblasts. AS602801 also inhibited the self-renewal and tumor-initiating capacity of cancer stem cells surviving AS602801 treatment. Cancer stem cells in established xenograft tumors were reduced by systemic administration of AS602801 at a dose and schedule that did not adversely affect the health of the tumor-bearing mice. These findings suggest AS602801 is a promising anti-cancer stem cell agent, and further investigation of the utility of AS602801 in the treatment of cancer seems warranted.

Time-staggered inhibition of JNK effectively sensitizes chemoresistant ovarian cancer cells to cisplatin and paclitaxel.

Ovarian cancer is the most lethal gynecological malignancy, for which platinum- and taxane-based chemotherapy plays a major role. Chemoresistance of ovarian cancer poses a major obstacle to the successful management of this devastating disease; however, effective measures to overcome platinum and taxane resistance are yet to be established. In the present study, while investigating the mechanism underlying the chemoresistance of ovarian cancer, we found that JNK may have a key role in the resistance of ovarian cancer cells to cisplatin and paclitaxel. Importantly, whereas simultaneous application of a JNK inhibitor and either of the chemotherapeutic agents had contrasting effects for cisplatin (enhanced cytotoxicity) and paclitaxel (decreased cytotoxicity), JNK inhibitor treatment prior to chemotherapeutic agent application invariably enhanced the cytotoxicity of both drugs, suggesting that the basal JNK activity is commonly involved in the chemoresistance of ovarian cancer cells to cisplatin and paclitaxel in contrast to drug‑induced JNK activity which may have different roles for these two drugs. Furthermore, we confirmed using non-transformed human and rodent fibroblasts that sequential application of the JNK inhibitor and the chemotherapeutic agents did not augment their toxicity. Thus, our findings highlight for the first time the possible differential roles of the basal and induced JNK activities in the chemoresistance of ovarian cancer cells and also suggest that time‑staggered JNK inhibition may be a rational and promising strategy to overcome the resistance of ovarian cancer to platinum- and taxane-based chemotherapy.

Licochalcone A specifically induces cell death in glioma stem cells via mitochondrial dysfunction

Glioblastoma multiforme is the most malignant primary intrinsic brain tumor. Glioma stem cells (GSCs) are associated with chemoradiotherapy resistance and the recurrence of glioblastomas after conventional therapy. The targeting of GSCs is potentially an effective treatment for the long‐term survival of glioblastoma patients. Licochalcone A, a natural chalconoid from licorice root, exerts anticancer effects; however, its effect on GSCs remains unknown. We found that Licochalcone A induced massive caspase‐dependent death in GSCs but not in differentiated GSCs nor normal somatic and neural stem cells. Prior to cell death, Licochalcone A caused mitochondrial fragmentation and reduced the membrane potential and ATP production in GSCs. Thus, Licochalcone A induces mitochondrial dysfunction and shows promise as an anticancer stem cell drug.

Involvement of GLUT1-mediated glucose transport and metabolism in gefitinib resistance of non-small-cell lung cancer cells

Use of epidermal growth factor receptor (EGFR) inhibitors represented by gefitinib and erlotinib has become the standard of treatment for non-small-cell lung cancers (NSCLCs) with activating EGFR mutations. However, the majority of NSCLCs, which overexpress EGFR without such mutations, are resistant to EGFR inhibitors, and the mechanism(s) behind such primary resistance of NSCLCs without activating EGFR mutations to EGFR inhibitors still remains poorly understood. Here in this study, we show that glucose metabolism mediated by GLUT1, a facilitative glucose transporter, is involved in gefitinib resistance of NSCLC cells. We found that GLUT1 expression and glucose uptake were increased in resistant NSCLC cells after gefitinib treatment and that genetic as well as pharmacological inhibition of GLUT1 sensitized not only NSCLC cells with primary resistance but also those with acquired resistance to gefitinib. In vivo, the combination of systemic gefitinib and a GLUT1 inhibitor, both of which failed to inhibit tumor growth when administered alone, significantly inhibited the growth of xenograft tumors formed by the implantation of NSCLC cells with wild-type EGFR (wt-EGFR). Since our data indicated that GLUT1 was similarly involved in erlotinib resistance, our findings suggest that the activity of GLUT1-mediated glucose metabolism could be a critical determinant for the sensitivity of NSCLC cells to EGFR inhibitors and that concurrent GLUT1 inhibition may therefore be a mechanism-based approach to treating NSCLCs resistant to EGFR inhibitors, including those with wt-EGFR.

A Small-molecule Kinase Inhibitor, CEP-1347, Inhibits Survivin Expression and Sensitizes Ovarian Cancer Stem Cells to Paclitaxel

Background: Chemoresistance of cancer stem cells (CSCs) is considered a major cause of post-treatment recurrence that negatively impacts the prognosis of patients with ovarian cancer. Materials and Methods: Using CSCs derived from two different ovarian cancer cell lines, we searched for molecules implicated in the chemoresistance of ovarian CSCs and also drugs with which to target those molecules. Results: Knockdown of survivin overexpressed in ovarian CSCs resulted in increased sensitivity to paclitaxel. Treatment at clinically relevant concentrations with CEP-1347, a mixed lineage kinase inhibitor with a known safety profile in humans, reduced survivin expression in ovarian CSCs and sensitized them to paclitaxel. Conclusion: Survivin overexpression plays a key role in the chemoresistance of ovarian CSCs. Introduction of CEP-1347, which targets survivin expression in ovarian CSCs, as a chemosensitizer for conventional ovarian cancer chemotherapy may serve as a rational and feasible approach for better management of ovarian cancer.

AS602801, an Anti-Cancer Stem Cell Drug Candidate, Suppresses Gap-junction Communication Between Lung Cancer Stem Cells and Astrocytes

Background/Aim: Cancer stem cells (CSCs) are associated with tumorigenesis, recurrence, and metastasis. Cell–cell communication via gap junctions (GJs) between metastatic cancer cells and astrocytes is necessary for brain metastasis. Agents targeting communication between CSCs and astrocytes are expected to suppress brain metastasis. Materials and Methods: Using the A549 CSC, a cancer stem-like cell derived from A549, we examined the effect of AS602801, an anti-cancer stem cell agent whose safety has been confirmed in a phase 2 clinical trial, on GJ communication and connexin expression using a dye-transfer assay and immunoblot analysis, respectively. Results: AS602801 specifically suppressed cell–cell communication in A549 CSCs without any suppression of GJ communication in astrocytes; it also decreased the expression of connexin 43, a constituent of GJs, in A549 CSCs. Conclusion: The anti-cancer stem cell agent, AS602801, is a potential drug candidate against brain metastasis.

Antitumor activity of gemcitabine against high-grade meningioma in vitro and in vivo

Currently, there is no established therapeutic option for high-grade meningioma recurring after surgery and radiotherapy, and few chemotherapeutic agents are in development for the treatment of high-grade meningioma. Here in this study, we screened a panel of chemotherapeutic agents for their possible antitumor activity in high-grade meningioma and discovered that high-grade meningioma cells show a preferential sensitivity to antimetabolites, in particular, to gemcitabine. In vitro, gemcitabine inhibited the growth of high-grade meningioma cells effectively by inducing S-phase arrest and apoptotic cell death. In vivo, systemic gemcitabine chemotherapy suppressed not only tumor initiation but also inhibited the growth and achieved a long-term control of established tumors in xenograft models of high-grade meningioma. Histological analysis indicated that systemic gemcitabine blocks cell cycle progression and promotes apoptotic cell death in tumor cells in vivo. Together, our data demonstrate that gemcitabine exerts potent antitumor activity against high-grade meningioma through cytostatic and cytotoxic mechanisms. We therefore propose gemcitabine is a promising chemotherapeutic agent that warrants further investigation as a treatment option for high-grade meningioma.Currently, there is no established therapeutic option for high-grade meningioma recurring after surgery and radiotherapy, and few chemotherapeutic agents are in development for the treatment of high-grade meningioma. Here in this study, we screened a panel of chemotherapeutic agents for their possible antitumor activity in high-grade meningioma and discovered that high-grade meningioma cells show a preferential sensitivity to antimetabolites, in particular, to gemcitabine. In vitro, gemcitabine inhibited the growth of high-grade meningioma cells effectively by inducing S-phase arrest and apoptotic cell death. In vivo, systemic gemcitabine chemotherapy suppressed not only tumor initiation but also inhibited the growth and achieved a long-term control of established tumors in xenograft models of high-grade meningioma. Histological analysis indicated that systemic gemcitabine blocks cell cycle progression and promotes apoptotic cell death in tumor cells in vivo. Together, our data demonstrate that gemcitabine exerts potent antitumor activity against high-grade meningioma through cytostatic and cytotoxic mechanisms. We therefore propose gemcitabine is a promising chemotherapeutic agent that warrants further investigation as a treatment option for high-grade meningioma.

Repositioning CEP-1347, a chemical agent originally developed for the treatment of Parkinson’s disease, as an anti-cancer stem cell drug

CEP-1347 is a mixed lineage kinase inhibitor tested in a large-scale phase 2/3 clinical trial in early Parkinson’s disease, in which its safety and tolerability, but nevertheless not efficacy, was demonstrated. Here we identify by drug repositioning CEP-1347 as a potential anti-cancer stem cell drug. In vitro, CEP-1347 efficiently induced differentiation and inhibited the self-renewal and tumor-initiating capacities of human cancer stem cells from glioblastoma as well as from pancreatic and ovarian cancers at clinically-relevant concentrations, without impairing the viability of normal fibroblasts and neural stem cells. In vivo, a 10-day systemic administration of CEP-1347 at a dose that was less than 1/10 the mouse equivalent of the dose safely given to humans for 2 years was sufficient to effectively reduce tumor-initiating cancer stem cells within established tumors in mice. Furthermore, the same treatment protocol significantly extended the survival of mice receiving orthotopic implantation of glioma stem cells. Together, our findings suggest that CEP-1347 is a promising candidate for cancer stem cell-targeting therapy and that further clinical and preclinical studies are warranted to evaluate its efficacy in cancer treatment.