Ryoko Kuniyoshi

Circulating tumor cells as a surrogate marker for determining response to chemotherapy in Japanese patients with metastatic colorectal cancer

The purpose of this study was to investigate the potential of circulating tumor cells (CTC) as a surrogate marker of the clinical outcome in metastatic colorectal cancer (mCRC) patients in order to identify Japanese patients responsive to oxaliplatin‐based chemotherapy. Between January 2007 and April 2008, 64 patients with mCRC were enrolled in this prospective study. The treatment regimen was oxaliplatin‐based chemotherapy. Collection of CTC from whole blood was performed at baseline and at 2 and 8–12 weeks after initiation of chemotherapy. Isolation and enumeration of CTC was performed using immunomagnetics. Patients with ≥3 CTC at baseline and at 2 and 8–12 weeks had a shorter median progression‐free survival (8.5, 7.3 and 1.9 months, respectively) than those with <3 CTC (9.7, 10.4 and 9.1 months, respectively) (log‐rank test: P = 0.047, P < 0.001 and P < 0.001, respectively). Patients with ≥3 CTC at 2 and 8–12 weeks had a shorter median overall survival (10.2 and 4.1 months, respectively) than those with <3 CTC (29.1 and 29.1 months, respectively) (P < 0.001 and P = 0.001, respectively). A spurious early rise in carcinoembryonic antigen level was observed in 11 patients showing a partial response. In contrast, no rise in early CTC level was observed among responders. Our data support the clinical utility of CTC enumeration in improving our ability to accurately assess treatment benefit and in expediting the identification of effective treatment regimens for individual Japanese patients. (Cancer Sci 2011; 102: 1188–1192)

Autophagy and autophagic cell death are next targets for elimination of the resistance to tyrosine kinase inhibitors

Autophagy, a cellular degradation system has been demonstrated in some hematopoietic malignant cell lines, but there is much still remaining to be known about its role and the mechanisms. We observed the excessive autophagy in chronic myelogenous leukemia (CML) cell line, K562, associated with treatment of 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), which can induce K562 cells to differentiate into megakaryocytic lineage. Confocal microscopic analysis demonstrated that autophagic cells did not express a megakaryocyte marker, the CD41 molecule, indicating that the autophagy was independent of megakaryocytic differentiation. After remarkable autophagic degradation, the cells finally underwent autophagic cell death (APCD). On the other hand, a block of TPA‐induced autophagy by chloroquine rapidly promoted cell death that was not APCD. This result suggested that autophagy regulated two mechanisms in K562 cells: both the cell survival system and APCD. To confirm that autophagy regulates the cell survival system in K562 cells, imatinib was used to induce cell death in K562 cells. Autophagy has not been considered during imatinib treatment; nonetheless, co‐treatment with imatinib and chloroquine markedly enhanced imatinib‐induced cell death, compared to K562 cells treated only with imatinib. Furthermore, imatinib‐resistant cell lines, BaF3/T315I and BaF3/E255K, also underwent cell death by co‐treatment with imatinib and chloroquine. From these data, we concluded that autophagy is deeply related to the cell survival system and that inhibition of autophagy accelerates TPA‐ or imatinib‐induced cell death. The block of autophagy could be a new strategy in the treatment of CML. (Cancer Sci 2008; 99: 2200–2208)

Identification of CD20 C-Terminal Deletion Mutations Associated with Loss of CD20 Expression in Non-Hodgkin's Lymphoma

Purpose: Rituximab is commonly incorporated into CD20-positive B-cell lymphoma therapy to improve response and prognosis. With increasing use, resistance to rituximab is a continuing concern, but CD20 mutation as a cause of resistance has not previously been reported. Experimental Design: Freshly collected lymphoma cells from 50 patients with previously untreated or relapsed/resistant non-Hodgkins B-cell lymphomas (diffuse large B cell, n = 22; follicular, n = 7; mucosa associated lymphoid tissue, n = 16; chronic lymphocytic leukemia, n = 2; small lymphocytic lymphoma, n = 1; lymphoplasmacytic, n = 1; mantle cell lymphoma, n = 1) were assessed for CD20 expression by flow cytometry, and CD20 gene sequencing was done on extracted DNA. Results: CD20 mutations were found in 11 (22.0%) of 50 patients and could be grouped as C-terminal deletion (8.0%), early termination (10.0%), and extracellular domain (2.0%) or transmembrane domain (2.0%) mutations. The mean fluorescence intensity of CD20 on fresh lymphoma cells was significantly lower for the C-terminal deletion mutation [3.26; 95% confidence interval (95% CI), 0.09-6.89] compared with wild type (30.8; 95% CI, 22.4-39.2; P < 0.05). In contrast, early termination mutations did not show significant differences in CD20 expression compared with wild type (19.5; 95% CI, 10.7-28.4; P > 0.05). Conclusions: It is possible that C-terminal deletion mutations of CD20 may be related to relapse/resistance after rituximab therapy. These mutations should be examined in patients showing progression of disease after partial remission.

Circulating endothelial progenitors and CXCR4‐positive circulating endothelial cells are predictive markers for bevacizumab

Bevacizumab plus chemotherapy is a standard option in the treatment of metastatic colorectal cancer (mCRC). The aim of this study was to investigate the potential of circulating endothelial cell progenitors (CEPs) and phenotypical circulating endothelial cells (CECs) as surrogate markers of clinical outcome in mCRC patients to identify responders to bevacizumab in combination with chemotherapy.

High reproducible ADCC analysis revealed a competitive relation between ADCC and CDC and differences between FcγRllla polymorphism

The anti-CD20 chimeric monoclonal antibody rituximab mediates cytotoxicity in malignant B cells via multiple mechanisms, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and direct induction of apoptosis. To optimize treatment of non-Hodgkins lymphoma, a fuller understanding of these mechanisms and their relative contributions to clinical efficacy is required. Here, we report the characteristics of the mutual impact between ADCC and CDC, the two major effector functions through the Fc receptors. To compare ADCC induced under various conditions, we developed a highly reproducible method of estimating ADCC activity using immortalized effector cells. The set of the effector cells that we established was able to calculate net ADCC with high reproducibility by comparing the cytotoxicity of effector cells expressing exogeneous FcγRIIIa to those of mock effector cells. In addition, the different property of effector cells of two FcγRIIIa single-nucleotide polymorphisms (SNP) could be also evaluated in exactly identical background. ADCC assessment in the presence of human serum directly provided the evidence of the competitive interaction of ADCC and CDC. The inhibition of ADCC of effector cells having low affinity SNP of FcγRIIIa by active complement was more potent than those having high-affinity SNP at the rituximab-concentration comparable to the serum level obtained in patients. These findings could have a profound impact on optimization of the regimen of therapeutic antibodies and on the development of antibodies that will enhance effector function.

Continuous treatment of bestatin induces anti‐angiogenic property in endothelial cells

CD13/aminopeptidase‐N (CD13/APN) is an important regulator of angiogenesis where its expression on activated blood vessels is induced by angiogenic signals. A previous study demonstrated that angiogenesis is suppressed under the presence of high concentrations of aminopeptidase antagonists. However, the mechanisms underlying the inhibition of morphogenesis by aminopeptidase antagonists have not been elucidated. In this study, we have for the first time examined the effects of continuous treatment of therapeutic dose of aminopeptidase antagonists on vascular endothelial capillary‐like tube formation. In the antagonists tested, only bestatin significantly interfered in the capillary tube formation of primary endothelial cells (EC) after treatment for 72 h. Aminopeptidase analysis revealed that inhibitory activity of bestatin was not specific for CD13/APN, and the other inhibitors lacking anti‐angiogenic properties also inhibit cell‐surface aminopeptidase activity as well or more potently than bestatin, suggesting that the angiogenesis‐inhibitory effect of bestatin was not due to inhibition of CD13/APN activity at this concentration. To elucidate the influence of continuous treatment of bestatin on endothelial cells, we performed microarray analysis and revealed that 72‐h treatment of a pharmacokinetic dose of bestatin modulated the several angiogenesis‐related genes including vascular endothelial growth factor (VEGF). Northern blot analysis indicated that modulation of the VEGF gene became obvious after 48 h of treatment. Furthermore, knockdown of the VEGF gene by siRNA remarkably suppressed capillary tube formation and required a higher concentration of exogenous VEGF to reverse the capillary formation ability. These data suggested that bestatin decreases a reactivity of EC to angiogenesis stimuli, and it can be achieved by the regulation of angiogenesis‐related gene expression. (Cancer Sci 2007; 98: 364–372)

Sumoylation and Apoptosis

Apoptosis is a physiological mechanism to maintain human tissues or cells, and pathological dysfunction of this process explains some disease states or drug resistance. P53, mdm2, sumoylation, signal transduction, and transcriptional control are important in regulation in cancer cells. We focus on the mechanisms of apoptosis in cancer cells involving SUMO.

Abstract B118: Histone demethylase inhibitor, trans-2-phenylcyclopropylamine hydrochloride inhibits KDM3A and KDM5A activities, resulting in overcoming resistance against bortezomib.

Purpose: The proteasome inhibitor bortezomib has shown impressive clinical activity alone and in combination with conventional and other novel agents for the myeloma treatment. Although bortezomib is known to be a selective proteasome inhibitor, the downstream mechanisms of cytotoxicity and drug resistance remain to be understood yet. However, resistance to bortezomib as a single agent develops in the majority of patients, and activity in other malignancies has been less impressive. To overcome bortezomib resistance, we compared differential gene expression profiles of bortezomib-resistant and bortezomib-sensitive IM-9 myeloma cell lines in response to bortezomib, and expressions of KDM3A and 5A genes were significantly incresed. Methods: The differential gene expression profiles of bortezomib-resistant IM-9 and bortezomib-sensitive IM-9 multiple myeloma cell lines in response to bortezomib was performed using Affymetrix GeneChip. To confirm the results, real-time PCR and Western blot analysis were performed. bortezomib-resistant IM-9 cells were treated with or without bortezomib and/or histone demethylase inhibitor, trans-2-phenylcyclopropylamine hydrochloride (2-PCPA). Moreover, level of histone methylation was examined by western blot analysis. Results: At concentrations that effectively inhibited proteasome activity (maximum dose with 100nM), bortezomib induced cell death in bortezomib-sensitive IM-9 cells, but not in bortezomib-resistant IM-9. In comparison of differential gene expression profiles between bortezomib-resistant IM-9 and bortezomib-sensitive IM-9 cells, we showed overexpression of KDM3A and KDM5A, which are associated with chromatin-mediated reversible drug-tolerant state, Moreover, 2-PCPA overcomes bortezomib resistance in a dose-dependent manner. The histone demethylation at H3K4, H3K9 and H3K27 was suppressed with 2-PCPA. Conclusion: Histone lysine-specific demethylase inhibitor, 2-PCPA with bortezomib may be useful for overcoming bortezomib-resistance in myeloma cells by suppression of KDM3A and KDM5A activities. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B118. Citation Format: Yasuhito Terui, Ryoko Kuniyoshi, Akihiro Tomida, Kiyohiko Hatake. Histone demethylase inhibitor, trans-2-phenylcyclopropylamine hydrochloride inhibits KDM3A and KDM5A activities, resulting in overcoming resistance against bortezomib. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B118.

Abstract A183: Ex vivo imaging of EGF receptor‐mediated signal transduction for prediction of anti‐EGFR treatment efficacy

Objectives: EGFR is a promising treatment target in solid tumors. Two major types of anti‐EGFR agents have entered the clinical setting: antibodies and small‐molecule EGFR TKIs. EGFR‐TKI inhibits autophosphorylation of EGFR by binding to ATP binding site of tyrosine kinase domain of EGFR and interrupts the signal transduction. On the other hand, anti‐EGER therapeutic antibodies interrupt EGFR signaling by blocking ligand binding. In addition, as for antibodies, the effects of the ADCC are also expected. In this study, we aimed at developing a method to estimate the effectiveness of anti‐EGFR agents by analyzing these mechanisms using ex vivo imaging technique. Methods: To establish a method that confirms the inhibition effect of the EGFR‐signal transduction, we prepared fluorescent‐labeled ligand and developed the imaging strategy to visualize membrane binding and subsequent internalization of labeled EGF. In addition, fluorescent‐labeled NK‐based effector cells that have been established by introducing fcgr3a gene into a NK leukemia cell line were used for characterization of ADCC susceptibility of individual tumor cells. In our strategy, a continuous confocal laser scanning microscopy was used for tracing fluorescent‐labeled EGF or effector cells as well as tumor cell death. Results: Fluorescent probe bearing EGF was highly specific and potent in the binding and activation of the EGF receptor, being rapidly internalized into endosomes. Pretreatment with therapeutic anti‐EGFR antibody completely inhibited the ligand‐binding and subsequent events. Meanwhile, EGFR TKIs partially prevented endocytotic step. K‐ras mutation status did not significantly affect the ligand binding and internalization. The imaging analysis of the ADCC using primary colorectal cancer specimens revealed that cetuximab‐coated tumor cells were gradually damaged by direct interaction of effector cells. ADCC susceptibility of individual tumor cells has varied to some extent. Conclusion: We have established imaging methods of the early events of EGFR signaling pathway and ADCC reaction. This imaging method soffer the rapid reliable means that analyze the effect of the EGFR targeted agent to the individual tumor cells. By combining with already established prognostic factors such as K‐ras mutation status or Fc receptor polymorphism, our imaging analysis will propose novel strategy to predict an individual treatment prognosis more precisely. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A183.

Abstract B68: Ubiquitin E3 ligase, tripartite motif protein 68 (TRIM68) inhibits TCP‐1β function and can be a target of imatinib‐resistance

Background: Resistance to imatinib mesylate is a major problem in chronic myeloid leukaemia (CML) treatment. Most of the studies about the resistance have focused on point mutations in BCR/ABL. However, other mechanisms of resistance that do not imply mutations in BCR/ABL have been also described. The identification of new proteins induced by imatinib may lead to find the novel potent molecular targets in imatinib‐resistant CML. Methods: K562 cells were treated with or without 1 µM imatinib for 24 hours, and then differential display between them was performed. TRIM68 expression was examined by RT‐PCR, and in vivo ubiquitination or sumoylation assay was performed by transfection experiment and Western blot analysis. The substrates for TRIM68 were analyzed by mass spectorometry. Results: From the results of RNA differential display, we found that the expression of TRIM68 mRNA was increased when the K562 cells were treated with 1 µM imatinib for 24 hours. TRIM68 protein possesses a RING finger domain at its N‐terminal site. Since many RING‐finger proteins have been identified as E3 ligases for ubiquitination or sumoylation, we examined whether TRIM68 functions as an E3 ligase for ubiquitination or sumoylation. To examine the function of TRIM68 as an E3 ligase, wild type TRIM68 and a RING domain deletion mutant of TRIM68 (TRIM68/ΔR) genes were constructed into a mammalian expression vector and they were transfected into MCF7 cells. TRIM68 had auto‐ubiquitination activity but not autosumoylation activity in vivo assays. This suggests that TRIM68 is could be an ubiquitin E3 ligase but not sumo E3 ligase. Moreover, wild type TRIM68 promoted the whole ubiqutination in the cells, whereas TRIM68/ΔR prevented the ubiquitination inside of the cells. To identify the TRIM68‐interacting proteins, we transfected FLAG‐tagged wild type TRIM68 gene or B30.2/SPRY domain of TRIM68 gene into MCF7 cells, and immunoprecipitation with FLAG‐M2 agarose was performed and mass spectrometric analysis was performed. As the results, we identified the members of molecular chaperone T‐complex polypeptide 1 (TCP‐1) complex, TCP‐1β and heat shock protein 70 interacting with TRIM68 at the B30.2/SPRY domain. Then, we examined whether TCP‐1β is one of the substrates for TRIM68‐ related ubiqutination. TCP‐1β was ubiquitinated by wild type TRIM68, but not by TRIM68/ΔR. Furthermore, the ubiquitination of TCP‐1β was accumulated by the treatment with a proteasome inhibitor MG132. These suggested that TCP‐1β is one of the substrates for TRIM68. Conclusion: We found that TRIM68 is induced by the treatment with imatinib and functions as an ubiquitin E3 ligase. Furthermore, we identified that TCP‐1β is a substrate of TRIM68. TRIM68 may inhibit the function of TCP‐1β as a chaperone by ubiquitination and proteasome‐mediated degradation. TRIM68 could be a new target in the imatinib‐resistant CML. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B68.