Wei Ye

Critical Role of the Stress Chaperone GRP78/BiP in Tumor Proliferation, Survival, and Tumor Angiogenesis in Transgene-Induced Mammary Tumor Development

The unfolded protein response (UPR) is an evolutionarily conserved mechanism that activates both proapoptotic and survival pathways to allow eukaryotic cells to adapt to endoplasmic reticulum (ER) stress. Although the UPR has been implicated in tumorigenesis, its precise role in endogenous cancer remains unclear. A major UPR protective response is the induction of the ER chaperone GRP78/BiP, which is expressed at high levels in a variety of tumors and confers drug resistance in both proliferating and dormant cancer cells. To determine the physiologic role of GRP78 in in situ-generated tumor and the consequence of its suppression on normal organs, we used a genetic model of breast cancer in the Grp78 heterozygous mice where GRP78 expression level was reduced by about half, mimicking anti-GRP78 agents that achieve partial suppression of GRP78 expression. Here, we report that Grp78 heterozygosity has no effect on organ development or antibody production but prolongs the latency period and significantly impedes tumor growth. Our results reveal three major mechanisms mediated by GRP78 for cancer progression: enhancement of tumor cell proliferation, protection against apoptosis, and promotion of tumor angiogenesis. Importantly, although partial reduction of GRP78 in the Grp78 heterozygous mice substantially reduces the tumor microvessel density, it has no effect on vasculature of normal organs. Our findings establish that a key UPR target GRP78 is preferably required for pathophysiologic conditions, such as tumor proliferation, survival, and angiogenesis, underscoring its potential value as a novel therapeutic target for dual antitumor and antiangiogenesis activity.

Stromal Matrix Metalloproteinase-9 Regulates the Vascular Architecture in Neuroblastoma by Promoting Pericyte Recruitment

Advanced stages of neuroblastoma show increased expression of matrix metalloproteinases MMP-2 and MMP-9 (Y. Sugiura et al., Cancer Res., 58: 2209–2216, 1998) that have been implicated in many steps of tumor progression, suggesting that they play a contributory role. Using pharmacological and genetic approaches, we have examined the role of these MMPs in progression of SK-N-BE (2).10 human neuroblastoma tumors orthotopically xenotransplanted into immunodeficient mice. Mice treated with Prinomastat, a synthetic inhibitor of MMPs, showed an inhibition of tumor cell proliferation in implanted tumors and a prolonged survival (50 versus 39 days in control group, P < 0.035). Treatment with Prinomastat did not affect formation of liver metastases (P = 0.52) but inhibited intravascular colonization by the tumor cells in the lung by 73.8% (P = 0.03) and angiogenesis in both primary tumors and experimental liver metastases. The primary tumors from Prinomastat-treated mice showed a 39.3% reduction in endothelial area detected by PECAM/CD31 staining in tumor sections (P < 0.001), primarily due to the presence of smaller vessels (P = 0.004). MMP-2 is expressed by neuroblastoma tumor cells and stromal cells, whereas MMP-9 is exclusively expressed by stromal cells, particularly vascular cells. To examine the contribution of MMP-9 to tumor angiogenesis, we generated RAG1/MMP-9 double-deficient mice. We observed a significant inhibition of angiogenesis in the immunodeficient RAG1/MMP-9 double-deficient mice orthotopically implanted with tumor cells (P = 0.043) or implanted s.c. with a mixture of tumor cells and Matrigel (P < 0.001). Using an FITC-labeled lectin, we demonstrated an inhibition in the architecture of the tumor vasculature in MMP-9-deficient mice, resulting in fewer and smaller blood vessels. These changes were associated with a 48% decrease in pericytes present along microvessels. Taken together, the data demonstrate that in neuroblastoma, stromally derived MMP-9 contributes to angiogenesis by promoting blood vessel morphogenesis and pericyte recruitment.

DNA methylation profiles in diffuse large B-cell lymphoma and their relationship to gene expression status

In an initial epigenetic characterization of diffuse large B-cell lymphoma (DLBCL), we evaluated the DNA methylation levels of over 500 CpG islands. Twelve CpG islands (AR, CDKN1C, DLC1, DRD2, GATA4, GDNF, GRIN2B, MTHFR, MYOD1, NEUROD1, ONECUT2 and TFAP2A) showed significant methylation in over 85% of tumors. Interestingly, the methylation levels of a CpG island proximal to FLJ21062 differed between the activated B-cell-like (ABC-DLBCL) and germinal center B-cell-like (GCB-DLBCL) subtypes. In addition, we compared the methylation and expression status of 67 genes proximal (within 500 bp) to the methylation assays. We frequently observed that hypermethylated CpG islands are proximal to genes that are expressed at low or undetectable levels in tumors. However, many of these same genes were also poorly expressed in DLBCL tumors where their cognate CpG islands were hypomethylated. Nevertheless, the proportional reductions in BNIP3, MGMT, RBP1, GATA4, IGSF4, CRABP1 and FLJ21062 expression with increasing methylation suggest that epigenetic processes strongly influence these genes. Lastly, the moderate expression of several genes proximal to hypermethylated CpG tracts suggests that DNA methylation assays are not always accurate predictors of gene silencing. Overall, further investigation of the highlighted CpG islands as potential clinical biomarkers is warranted.

A Critical Role for GRP78/BiP in the Tumor Microenvironment for Neovascularization during Tumor Growth and Metastasis

Glucose-regulated protein 78 (GRP78)/BiP is a multifunctional protein which plays a major role in endoplasmic reticulum (ER) protein processing, protein quality control, maintaining ER homeostasis, and controlling cell signaling and viability. Previously, using a transgene-induced mammary tumor model, we showed that Grp78 heterozygosity impeded cancer growth through suppression of tumor cell proliferation and promotion of apoptosis and the Grp78(+/-) mice exhibited dramatic reduction (70%) in the microvessel density (MVD) of the endogenous mammary tumors, while having no effect on the MVD of normal organs. This observation suggests that GRP78 may critically regulate the function of the host vasculature within the tumor microenvironment. In this article, we interrogated the role of GRP78 in the tumor microenvironment. In mouse tumor models in which wild-type (WT), syngeneic mammary tumor cells were injected into the host, we showed that Grp78(+/-) mice suppressed tumor growth and angiogenesis during the early phase but not during the late phase of tumor growth. Growth of metastatic lesions of WT, syngeneic melanoma cells in the Grp78(+/-) mice was potently suppressed. We created conditional heterozygous knockout of GRP78 in the host endothelial cells and showed severe reduction of tumor angiogenesis and metastatic growth, with minimal effect on normal tissue MVD. Furthermore, knockdown of GRP78 expression in immortalized human endothelial cells showed that GRP78 is a critical mediator of angiogenesis by regulating cell proliferation, survival, and migration. Our findings suggest that concomitant use of current chemotherapeutic agents and novel therapies against GRP78 may offer a powerful dual approach to arrest cancer initiation, progression, and metastasis.

Identification of micrometastases in histologically negative lymph nodes of early-stage cervical cancer patients.

OBJECTIVE: Despite histologically negative lymph nodes, approximately 15% of patients with early-stage cervical cancer will develop recurrence. Micrometastases have been shown to be important in staging and treatment of breast cancers and melanoma and have been identified by polymerase chain reaction analysis in cervical cancers. This study sought to estimate the frequency of micrometastases identified by immunohistochemistry in histologically negative lymph nodes and compare this to other known risk factors for recurrence of cervical cancer. METHODS: Early-stage (stages IA2, IB1, and IB2) cervical cancer patients of all histologic subtypes were identified from the surgical logs of the Los Angeles County–University of Southern California Medical Center for the period 1994–2000. One hundred thirty-two patients had histologically negative lymph nodes. Immunohistochemical assay was performed on 3,106 lymph nodes by using antibodies against cytokeratins AE-1 and CAM 5.2 in combination according to standard protocols. The stained nodes were then evaluated for the presence of micrometastases and compared against the respective clinicopathologic information in each case. RESULTS: Micrometastases were detected in 19 of 132 (15%, 95% confidence interval [CI] 9%, 22%) patients, found in 29 of the 3,106 (0.9%) lymph nodes evaluated. Vascular space invasion was seen in 50 of 132 cases (38%, 95% CI 30%, 47%) and in 8 of 19 (42%, 95% CI 21%, 66%) cases with micrometastases. Surgical margins of the resected specimen were negative in 120 of 132 cases (91%, 95% CI 84%, 95%) and in 16 of 19 (84%, 95%CI 60%, 96%) of those cases with micrometastases. Micrometastases were seen most frequently in pelvic lymph nodes (25 of 29, 86%). Patients with more than 20 lymph nodes removed were more likely to demonstrate metastasis (P < .001). There was no statistically significant association between micrometastasis and vascular space invasion or tumor volume. CONCLUSION: Micrometastases are identifiable in histologically negative lymph nodes in 15% (95% CI 9%, 22%) of early-stage cancer patients, a frequency which approximates the recurrence rate for patients with negative nodes. In this series, patients with greater numbers of lymph nodes analyzed were more likely to have lymph node micrometastasis identified. There appears to be no relationship between tumor volume and the identification of micrometastases. Although micrometastases can be identified in histologically negative lymph nodes, their presence is not strongly associated with other known factors of cervical cancer recurrence. Further research is needed to determine whether the presence of lymph node micrometastases is associated with an unfavorable prognosis. LEVEL OF EVIDENCE: II-3

Predicting Recurrence and Progression of Noninvasive Papillary Bladder Cancer at Initial Presentation Based on Quantitative Gene Expression Profiles

BACKGROUND Currently, tumor grade is the best predictor of outcome at first presentation of noninvasive papillary (Ta) bladder cancer. However, reliable predictors of Ta tumor recurrence and progression for individual patients, which could optimize treatment and follow-up schedules based on specific tumor biology, are yet to be identified. OBJECTIVE To identify genes predictive for recurrence and progression in Ta bladder cancer at first presentation using a quantitative, pathway-specific approach. DESIGN, SETTING, AND PARTICIPANTS Retrospective study of patients with Ta G2/3 bladder tumors at initial presentation with three distinct clinical outcomes: absence of recurrence (n=16), recurrence without progression (n=16), and progression to carcinoma in situ or invasive disease (n=16). MEASUREMENTS Expressions of 24 genes that feature in relevant pathways that are deregulated in bladder cancer were quantified by real-time polymerase chain reaction on tumor biopsies from the patients at initial presentation. RESULTS AND LIMITATIONS CCND3 (p=0.003) and HRAS (p=0.01) were predictive for recurrence by univariate analysis. In a multivariable model based on CCND3 expression, sensitivity and specificity for recurrence were 97% and 63%, respectively. HRAS (p<0.001), E2F1 (p=0.017), BIRC5/Survivin (p=0.038), and VEGFR2 (p=0.047) were predictive for progression by univariate analysis. Multivariable analysis based on HRAS, VEGFR2, and VEGF identified progression with 81% sensitivity and 94% specificity. Since this is a small retrospective study using medium-throughput profiling, larger confirmatory studies are needed. CONCLUSIONS Gene expression profiling across relevant cancer pathways appears to be a promising approach for Ta bladder tumor outcome prediction at initial diagnosis. These results could help differentiate between patients who need aggressive versus expectant management.

Alum with Interleukin-12 Augments Immunity to a Melanoma Peptide Vaccine: Correlation with Time to Relapse in Patients with Resected High-Risk Disease

Purpose: We attempted to augment immunity to melanoma antigens using interleukin-12 (IL-12) with aluminum hydroxide (alum) for sustained release or granulocyte macrophage colony-stimulating factor (GM-CSF) added to a multipeptide vaccine. Experimental Design: Sixty patients with high-risk resected melanoma were randomized to receive melanoma peptides gp100209-217 (210M), MART-126-35 (27L), and tyrosinase368-376 (370D) with adjuvant Montanide ISA 51 and either IL-12 at 30 ng/kg with alum (group A), IL-12 at 100 ng/kg with alum (group B), or IL-12 at 30 ng/kg with 250 μg GM-CSF (group C). Results: Three patients had stage IIC (5%), 50 had stage III (83%), and 7 had stage IV (12%) melanoma. Most toxicities were grade 1/2 and resolved rapidly. Significant toxicity included grade 3 colitis and visual changes and grade 3 headache resolving after stopping IL-12 but continuing peptide vaccine. A higher rate of post-vaccine 6-month immune response to gp100 and MART-1 was observed in group A (15 of 19) or B (19 of 20) that received IL-12 plus alum versus group C with IL-12/GM-CSF (4 of 21; P < 0.001). Post-vaccine enzyme-linked immunospot response rates to peptide analogues in group B were higher than group A (P = 0.031 for gp100 and P = 0.010 for MART-1); both were higher than group C (P < 0.001 for gp100 and P < 0.026 for MART-1). With a median of 24 months of follow-up, 23 patients have relapsed. Post-vaccine immune response to MART-1 was associated with relapse-free survival (P = 0.012). Conclusions: IL-12 with alum augmented an immune response to melanoma antigens compared with IL-12 with GM-CSF. Immune response was associated with time to relapse.

Comparison of Iodine-123 Metaiodobenzylguanidine (MIBG) Scan and [18F]Fluorodeoxyglucose Positron Emission Tomography to Evaluate Response After Iodine-131 MIBG Therapy for Relapsed Neuroblastoma

PURPOSE Children with relapsed neuroblastoma have poor survival. It is crucial to have a reliable method for evaluating functional response to new therapies. In this study, we compared two functional imaging modalities for neuroblastoma: metaiodobenzylguanidine (MIBG) scan for uptake by the norepinephrine transporter and [(18)F]fluorodeoxyglucose positron emission tomography (FDG-PET) uptake for glucose metabolic activity. PATIENTS AND METHODS Patients enrolled onto a phase I study of sequential infusion of iodine-131 ((131)I) MIBG (NANT-2000-01) were eligible for inclusion if they had concomitant FDG-PET and MIBG scans. (131)I-MIBG therapy was administered on days 0 and 14. For each patient, we compared all lesions identified on concomitant FDG-PET and MIBG scans and gave scans a semiquantitative score. RESULTS The overall concordance of positive lesions on concomitant MIBG and FDG-PET scans was 39.6% when examining the 139 unique anatomic lesions. MIBG imaging was significantly more sensitive than FDG-PET overall and for the detection of bone lesions (P < .001). There was a trend for increased sensitivity of FDG-PET for detection of soft tissue lesions. Both modalities showed similar improvement in number of lesions identified from day 0 to day 56 scan and in semiquantitative scores that correlated with overall response. FDG-PET scans became completely negative more often than MIBG scans after treatment. CONCLUSION MIBG scan is significantly more sensitive for individual lesion detection in relapsed neuroblastoma than FDG-PET, though FDG-PET can sometimes play a complementary role, particularly in soft tissue lesions. Complete response by FDG-PET metabolic evaluation did not always correlate with complete response by MIBG uptake.

The Activity of Zoledronic Acid on Neuroblastoma Bone Metastasis Involves Inhibition of Osteoclasts and Tumor Cell Survival and Proliferation

Metastasis to the bone is seen in 56% of patients with neuroblastoma and contributes to morbidity and mortality. Using a murine model of bone invasion, we have reported previously that neuroblastoma cells invade the bone by activating osteoclasts. Here, we investigated the antitumoral and antiosteolytic activities of zoledronic acid, a bisphosphonate inhibitor of osteoclasts, in combination with cytotoxic chemotherapy in our model. We first show that zoledronic acid given at the same time (early prevention) or 2 weeks after tumor cell injection (late prevention) significantly prevented the formation of severe osteolytic lesions. It also prevented formation of these lesions when given 4 weeks after tumor cell injection (intervention) when combined with chemotherapy including cyclophosphamide and topotecan. The combination of zoledronic acid + cyclophosphamide/topotecan also significantly improved survival (P < 0.001). In mice treated with zoledronic acid, we observed a marked inhibition of osteoclasts inside the bone associated with a decrease in tumor cell proliferation and increase in tumor cell apoptosis. In vitro, zoledronic acid inhibited neuroblastoma cell proliferation and induced apoptosis, and these effects were significantly enhanced by the addition of 4-hydroxyperoxycyclophosphamide (4-HC). The proapoptotic effect of zoledronic acid and zoledronic acid in combination with 4-HC on tumor cells was associated with an increase in caspase-3 activity and a decrease in phosphorylated Bcl-2, Bcl-2, and Bcl-X(L) expression. Zoledronic acid inhibited the association of Ras with the plasma membrane and activation of c-Raf, Akt, and extracellular signal-regulated kinase 1/2. The data indicate that zoledronic acid, in addition to inhibiting osteoclasts, is active against tumor cells and suggest that zoledronic acid in combination with cytotoxic chemotherapy may be effective in children with neuroblastoma that has metastasized to the bone.

The relationship between proangiogenic gene expression levels in prostate cancer and their prognostic value for clinical outcomes

Androgens stimulate the expression of vascular endothelial growth factor (VEGF) through activation of hypoxia inducible factor (HIF). These genes play a major role in cancer angiogenesis. This study assesses the relationship among expression levels for the androgen receptor (AR), HIF1a, VEGF‐A, and VEGF‐C genes in human prostate cancer tissue and their impact on prostate cancer outcomes. It also examines the impact of pre‐operative androgen deprivation therapy (ADT) on the expression of these genes.