Naoko Seki

Induction of tumor-specific T cell immunity by anti-DR5 antibody therapy

Because tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells and plays a critical role in tumor surveillance, its receptor is an attractive target for antibody-mediated tumor therapy. Here we report that a monoclonal antibody (mAb) against the mouse TRAIL receptor, DR5, exhibited potent antitumor effects against TRAIL-sensitive tumor cells in vivo by recruiting Fc receptor–expressing innate immune cells, with no apparent systemic toxicity. Administration of the agonistic anti-DR5 mAb also significantly inhibited experimental and spontaneous tumor metastases. Notably, the anti-DR5 mAb-mediated tumor rejection by innate immune cells efficiently evoked tumor-specific T cell immunity that could also eradicate TRAIL-resistant variants. These results suggested that the antibody-based therapy targeting DR5 is an efficient strategy not only to eliminate TRAIL-sensitive tumor cells, but also to induce tumor-specific T cell memory that affords a long-term protection from tumor recurrence.

Tumor-Specific CTL Kill Murine Renal Cancer Cells Using Both Perforin and Fas Ligand-Mediated Lysis In Vitro, But Cause Tumor Regression In Vivo in the Absence of Perforin

Kidney cancer is a devastating disease; however, biological therapies have achieved some limited success. The murine renal cancer Renca has been used as a model for developing new preclinical approaches to the treatment of renal cell carcinoma. Successful cytokine-based approaches require CD8+ T cells, but the exact mechanisms by which T cells mediate therapeutic benefit have not been completely identified. After successful biological therapy of Renca in BALB/c mice, we generated CTLs in vitro using mixed lymphocyte tumor cultures. These CTL mediated tumor-specific H-2Kd-restricted lysis and production of IFN-γ, TNF-α, and Fas ligand (FasL) in response to Renca. CTL used both granule- and FasL-mediated mechanisms to lyse Renca, although granule-mediated killing was the predominant lytic mechanism in vitro. The cytokines IFN-γ and TNF-α increased the sensitivity of Renca cells to CTL lysis by both granule- and FasL-mediated death pathways. Adoptive transfer of these anti-Renca CTL into tumor-bearing mice cured most mice of established experimental pulmonary metastases, and successfully treated mice were immune to tumor rechallenge. Interestingly, we were able to establish Renca-specific CTL from mice gene targeted for perforin (pfp−/−) mice. Although these pfp−/− CTL showed reduced cytotoxic activity against Renca, their IFN-γ production in the presence of Renca targets was equivalent to that of wild-type CTL, and adoptive transfer of pfp−/− CTL was as efficient as wild-type CTL in causing regression of established Renca pulmonary metastases. Therefore, although granule-mediated killing is of paramount importance for CTL-mediated lysis in vitro, some major in vivo effector mechanisms clearly are independent of perforin.

HLA class-I-restricted and tumor-specific CTL in tumor-infiltrating lymphocytes of patients with gastric cancer

Immune recognition of human cancers except melanoma is not well understood at either the cellular or the molecular level. We demonstrate in this study the existence of HLA class‐I‐restricted and tumor‐specific CTL in IL‐2‐activated TIL (tumor‐infiltrating lymphocytes) of all 4 gastric cancer patients tested. We established HLA A2‐restricted and adenocarcinoma‐specific CTL in 2 HLA A0201+ patients, and HLA A2402‐restricted CTL recognizing both adenocarcinoma and squamous‐cell carcinomas (SCC) in the 2 remaining HLA A2402+ patients. Further, HLA A3101‐restricted and adenocarcinoma‐specific CTL were established in 1 of the 2 HLA A2402+ patients who had HLA A3101 allele. HLA A2‐, A2402‐ and A3101‐restricted CD8+ CTL clones were established from these parental CTL lines. The 2 HLA A2‐restricted CTL lines lysed 8 of 13 HLA A2+ adenocarcinoma cell lines established from different organs (stomach, colon, lung and breast) with different subtypes (HLA A0201, A0206 and A0207). The HLA A2‐restricted CTL line recognized 9 and 6 different HPLC fractions of peptides eluted from the HLA A0201+ breast and HLA A0201+ colon adenocarcinoma cell lines, respectively. Allele‐specific deletion of HLA A2 or A24 molecules was observed in some tumor lines that were not susceptible to lysis by the CTL lines. These results suggest that TIL of gastric cancer possess CTL recognizing different peptide antigens binding to different HLA‐A alleles that are widely expressed on adenocarcinomas and also, to some extent, on SCC from different organs. Int. J. Cancer 70:631–638, 1997.

Bortezomib sensitizes human esophageal squamous cell carcinoma cells to TRAIL-mediated apoptosis via activation of both extrinsic and intrinsic apoptosis pathways

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive human cancers, and novel treatment modalities are required. We investigated the therapeutic potential of the tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2L) in combination with the proteasome inhibitor bortezomib (Velcade) on human ESCC cell lines. Bortezomib enhanced the susceptibility to TRAIL in 12 of the 15 ESCC cell lines tested, although most showed low sensitivity to TRAIL as a single agent. The enhancement of TRAIL-induced apoptosis by bortezomib was caspase dependent. Increased processing of caspase-8 often accompanied enhancement of TRAIL-induced apoptosis by bortezomib. However, the increased cell surface expression of death receptors observed on bortezomib treatment did not seem to be crucial for this effect. For some ESCC, bortezomib treatment resulted in a more efficient recruitment of caspase-8 and the Fas-associated death domain to the death-inducing signaling complex. Additional downregulation of the cellular FLICE-inhibitory protein long isoform [c-FLIP(L)] could cooperate in the activation of the extrinsic pathway in some cases. For other ESCC, the crucial effect of bortezomib treatment seemed to be increased signaling via the intrinsic apoptotic pathway on subsequent exposure to TRAIL. Thus, bortezomib could sensitize ESCC to TRAIL apoptosis by multiple molecular mechanisms of action. Therefore, the combination of bortezomib and TRAIL might be a novel therapeutic strategy for ESCC patients who fail to respond to standard chemoradiotherapy that predominantly targets the mitochondrial apoptotic pathway. Mol Cancer Ther; 9(6); 1842–51. ©2010 AACR.

FOXP3 expression in tumor cells and tumor-infiltrating lymphocytes is associated with breast cancer prognosis

The forkhead box protein 3 (FOXP3) transcription factor is highly expressed in tumor cells as well as in regulatory T cells (Tregs). It plays a tumor-enhancing role in Tregs and suppresses carcinogenesis as a potent repressor of several oncogenes. The clinical prognostic value of FOXP3 expression has not yet been elucidated. In this study, immunohistochemistry was used to investigate the prognostic significance of FOXP3 expression in tumor cells and tumor-infiltrating lymphocytes (TILs) in breast cancer patients. Of the 100 tumor specimens obtained from primary invasive breast carcinoma, 63 and 57% were evaluated as FOXP3+ tumor cells and as being highly infiltrated by FOXP3+ lymphocytes, respectively. Although FOXP3 expression in tumor cells was of no prognostic significance, FOXP3+ lymphocytes were significantly associated with poor overall survival (OS) (n=98, log-rank test P=0.008). FOXP3 exhibited a heterogeneous subcellular localization in tumor cells (cytoplasm, 31%; nucleus, 26%; both, 6%) and, although cytoplasmic FOXP3 was associated with poor OS (P= 0.058), nuclear FOXP3 demonstrated a significant association with improved OS (P=0.016). Furthermore, when patients were grouped according to their expression of tumor cytoplasmic FOXP3 and lymphocyte FOXP3, there were notable differences in the Kaplan-Meier curves for OS (P<0.001), with a high infiltration of FOXP3+ lymphocytes accompanied by a cytoplasmic FOXP3+ tumor being the most detrimental phenotype. These findings indicated that FOXP3 expression in lymphocytes as well as in tumor cells may be a prognostic marker for breast cancer. FOXP3 in tumor cells may have distinct biological activities and prognostic values according to its localization, which may help establish appropriate cancer treatments.

Tricin inhibits proliferation of human hepatic stellate cells in vitro by blocking tyrosine phosphorylation of PDGF receptor and its signaling pathways

4′,5,7‐Trihydroxy‐3′,5′‐dimethoxyflavone (Tricin), a naturally occurring flavone, has anti‐inflammatory potential and exhibits diverse biological activities including antigrowth activity in several human cancer cell lines and cancer chemopreventive effects in the gastrointestinal tract of mice. The present study aimed to investigate the biological actions of tricin on hepatic stellate cells (HSCs) in vitro, exploring its potential as a treatment of liver fibrosis, since HSC proliferation is closely related to the progression of hepatic fibrogenesis in chronic liver diseases leading to irreversible liver cirrhosis and hepatocellular carcinoma. Tricin inhibited platelet‐derived growth factor (PDGF)‐BB‐induced cell proliferation by blocking cell cycle progression and cell migration in the human HSC line LI90 and culture‐activated HSCs. It also reduced the phosphorylation of PDGF receptor β and the downstream signaling molecules ERK1/2 and Akt, which might be due to its tyrosine kinase inhibitor properties rather than inhibition of the direct binding between PDGF‐BB and its receptor. Our findings suggest that tricin might be beneficial in HSC‐targeting therapeutic or chemopreventive applications for hepatic fibrosis. J. Cell. Biochem. 113: 2346–2355, 2012.

Characterization of IL-2-activated TILs and their use in intrapericardial immunotherapy in malignant pericardial effusion

Pericardial effusion (PE) and cardiac tamponade caused by malignant pericarditis are critical conditions in cancer patients, which still lack a recommended protocol for their long-term management. Percutaneous pericardiocentesis and simple drainage are commonly performed as the initial treatment. The aims of this study were to investigate the presence of cytotoxic T lymphocytes (CTLs) in malignant PE and to determine the clinical response to administering autologous tumor-infiltrating lymphocytes (TILs) into the pericardial cavity. Initially, we identified human lymphocyte antigen class-I-restricted and tumor-specific CTLs within the interleukin-2 (IL-2)-activated TILs in PEs from four patients, on the basis of interferon-γ production and lactate dehydrogenase-release assays. Clinically we observed favorable responses to the pericardial transfer of IL-2-activated autologous TILs in four patients: one male with advanced esophageal cancer, one female with recurrent lung cancer and two females with recurrent breast cancer, respectively. Autologous TILs from PEs were expanded in vitro with IL-2, characterized for CD3, CD4 and CD8 markers, checked for contamination and then infused into the patient’s pericardial space through a catheter. This was repeated biweekly. After treatment, there were no signs of recurrence of PE in either case, as determined by radiography, echocardiography and computed tomography. The only adverse effects seen were grade 1 fevers. These results suggested that intrapericardial cellular immunotherapy with autologous TILs could be a safe and effective treatment for controlling malignant pericarditis with associated cardiac tamponade, and that tumor-specific CTLs present in malignant PE might be important for tumor rejection.

Predictive factors of the tumor immunological microenvironment for long-term follow-up in early stage breast cancer

The aim of this research was to investigate the correlation of immunologic factors in the tumor environment of breast cancer, using immunohistological staining to evaluate the expression of programmed death 1/programmed death ligand 1 (PD‐1/PD‐L1), phosphatase and tensin homolog (PTEN), tumor infiltrating lymphocytes (TILs), and macrophages, and to analyze the association between the immunologic factors and clinical outcome for patients with early stage breast cancer (EBC). A total of 97 EBC patients who underwent standard surgery were investigated. Expression of PD‐1/PD‐L1 and PTEN and the density of CD3+ TILs, CD8+ TILs, and CD163+ macrophages were evaluated by immunohistochemical analysis. The association between the immunologic factors and clinical outcome was statistically analyzed. The density of CD3+ TILs, CD8+ TILs, and CD163+ macrophages and non‐expression of PTEN was significantly higher in cases of triple negative breast cancer. CD8+ TIL density and CD8+/PD‐L1+ expression were predictive factors for disease‐free survival and overall survival (OS). Human epidermal growth factor 2 (HER2)‐positive patients with PTEN expression and luminal/HER2‐negative patients without PD‐L1 expression had significantly longer OS compared to patients without PTEN expression (P = 0.049) and with PD‐L1 expression (P = 0.036), respectively. Furthermore, patients with PD‐L1+/CD8+ expression had worse median progression‐free survival (P = 0.022) and median OS (P = 0.037) compared with patients without PD‐L1+/CD8+ expression. The CD3+ TILs, CD8+ TILs, and CD163+ macrophages were shown to infiltrate the tumor area of EBC. In particular, triple negative breast cancer had a higher rate of TIL infiltration within the tumor environment. Expression of PTEN and lack of PD‐L1 expression were associated with favorable survival in HER2‐positive and luminal/HER2‐negative EBC patients, respectively. The PD‐L1 expression combined with CD8+ density was significantly associated with an aggressive clinical outcome.

CD44v3+/CD24- cells possess cancer stem cell-like properties in human oral squamous cell carcinoma.

Cancer stem cells (CSCs) or cancer stem cell-like cells (CSC-LCs) are a minority population of cells that relate to tumor progression, metastasis and drug resistance. To identify CSC-LCs in oral squamous cell carcinoma (OSCC), we used two OSCC cell lines, SAS and OSC20, and cell surface markers, CD44v3 and CD24. In addition, we examined CD44v3 and CD24 expression immunohistochemically and evaluated the relationship between the expression and clinicopathological parameters in 50 OSCC tissues. In SAS and OSC20, CD44v3+/CD24− cells showed a higher sphere forming ability than the other fractions, i.e., CD44v3+/CD24+, CD44v3−/CD24− and CD44v3−/CD24+ cells. The proportion of CD44v3+/CD24− cells in SAS and OSC20 was 10.7 and 24.1%, respectively. Regarding SAS, CD44v3+/CD24− cells also showed a higher drug resistance for CDDP, 5-FU and cetuximab and expressed higher mRNA levels of CSC property-related genes than the other cell fractions. The tumorigenicity of CD44v3+/CD24− cells was not significantly different from the other fractions in SAS. An immunohistochemical study revealed a significant correlation between CD44v3 expression in the invasive portion and lymph node metastasis. Kaplan Meier analysis revealed cases with CD44v3 expression in the invasive portion tended to show poor overall survival (OS) compared with those without CD44v3, and there was a significant difference in OS between CD44v3+/CD24− and CD44v3−/CD24− immunophenotypes in the invasive portion. In conclusion, the results suggest that the CD44v3+/CD24− cell population displays CSC-LC properties in a human OSCC cell line. Additionally, we present evidence that CD44v3 immunoexpression and CD44v3+/CD24− immunophenotypes could give prognostic information associated with unfavorable clinical outcomes.

YB-1 prevents apoptosis via the mTOR/STAT3 pathway in HER-2-overexpressing breast cancer cells

Evaluation of: Lee C, Dhillon J, Wang MY et al.: Targeting YB-1 in HER-2 overexpressing breast cancer cells induces apoptosis via the mTOR/STAT3 pathway and suppresses tumor growth in mice. Cancer Res. 68 (21), 8661-8666 (2008). The transcription factor Y-box binding protein (YB)-1 is highly expressed in breast cancer cells and is strongly linked with breast cancer patient prognosis. In this paper, siRNA knockdown of YB-1 was used to investigate breast cancer cell proliferation. Six breast cancer cell lines that either overexpress HER-2 or were triple negative demonstrated growth inhibition following YB-1 knockdown. In particular, YB-1 knockdown induced apoptosis in BT-474-m1 and Au565 cells. Knockdown of YB-1 also decreased phosphorylation of STAT3S727, ERK1/2T202/Y204, mTORS2448 and total mTOR expression. When STAT3 was knocked down by siSTAT3, apoptosis was induced and constitutively active phosphorylated STAT3 was found to rescue YB-1-induced apoptosis. Furthermore, YB-1 knockdown remarkably suppressed colony formation in a soft agar assay, while delayed tumor formation was observed in mice. YB-1 knockdown inhibited cell growth and it is thought to involve induction of apoptosis via the mTOR/STAT3 intracellular signaling pathway. YB-1 is a promising molecular target for HER-2-overexpressing or triple-negative breast cancer cells.