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Immune checkpoint blockade involves the targeted antagonism of immunosuppressive interactions between antigen-presenting cells and/or tumor cells and effector T cells. Blockade of B7-H1, also known as programmed death-ligand 1 (PD-L1), prevents the ligation of inhibitory PD-L1 molecules to programmed cell death receptor 1 (PD-1) on T cells, engendering a potentiated response of tumor-specific T cells against tumor cells. In a Cancer Research article, Hirano and colleagues showed that T-cell–mediated tumor immunity becomes impaired when tumor cells interact with T cells via PD-L1 in the mouse tumor microenvironment. They showed that targeting PD-L1 or PD-1 with mAbs increased tumor cell lysis by T cells and suggested that tumor PD-L1 forms a “shield” preventing tumor cell lysis. Alongside other original mouse and human studies, this work generated scientific rationales for a new generation of cancer treatment focused on targeting the inhibitory PD-1/PD-L1 signaling pathway in the tumor microenvironment. See related article by Hirano and colleagues, Cancer Res 2005;65: 1089–96

Blood levels of acute-phase protein α

Mitochondrial DNA (mtDNA) mutations and polymorphisms contribute to many complex diseases, including cancer. Using a unique mouse model that contains nDNA from one mouse strain and homoplasmic mitochondrial haplotypes from different mouse strain(s)—designated Mitochondrial Nuclear Exchange (MNX)—we showed that mtDNA could alter mammary tumor metastasis. Because retrograde and anterograde communication exists between the nuclear and mitochondrial genomes, we hypothesized that there are differential mtDNA-driven changes in nuclear (n)DNA expression and DNA methylation. Genome-wide nDNA methylation and gene expression were measured in harvested brain tissue from paired wild-type and MNX mice. Selective differential DNA methylation and gene expression were observed between strains having identical nDNA, but different mtDNA. These observations provide insights into how mtDNA could be altering epigenetic regulation and thereby contribute to the pathogenesis of metastasis. Cancer Res; 77(22); 6202–14. ©2017 AACR.

Dendritic cell (DC)-based vaccine strategies aimed at targeting cancer stem–like cells (CSC) may be most efficacious if deployed in the adjuvant setting. In this study, we offer preclinical evidence that this is the case for a CSC-DC vaccine as tested in murine models of SCC7 squamous cell cancer and D5 melanoma. Vaccination of mice with an ALDHhigh SCC7 CSC-DC vaccine after surgical excision of established SCC7 tumors reduced local tumor relapse and prolonged host survival. This effect was augmented significantly by simultaneous administration of anti-PD-L1, an immune checkpoint inhibitor. In the minimal disease setting of D5 melanoma, treatment of mice with ALDHhigh CSC-DC vaccination inhibited primary tumor growth, reduced spontaneous lung metastases, and increased host survival. In this setting, CCR10 and its ligands were downregulated on ALDHhigh D5 CSCs and in lung tissues, respectively, after vaccination with ALDHhigh D5 CSC-DC. RNAi-mediated attenuation of CCR10 blocked tumor cell migration in vitro and metastasis in vivo. T cells harvested from mice vaccinated with ALDHhigh D5 CSC-DC selectively killed ALDHhigh D5 CSCs, with additional evidence of humoral immunologic engagement and a reduction in ALDHhigh cells in residual tumors. Overall, our results offered a preclinical proof of concept for the use of ALDHhigh CSC-DC vaccines in the adjuvant setting to more effectively limit local tumor recurrence and spontaneous pulmonary metastasis, as compared with traditional DC vaccines, with increased host survival further accentuated by simultaneous PD-L1 blockade. Cancer Res; 76(16); 4661–72. ©2016 AACR.

Nonlethal exposure to ionizing radiation (IR) is a public concern due to its known carcinogenic effects. Although latency periods for IR-induced neoplasms are relatively long, the ability to detect cancer as early as possible is highly advantageous for effective therapeutic intervention. Therefore, we hypothesized that metabolites in the urine from mice exposed to total body radiation (TBI) would predict for the presence of cancer before a palpable mass was detected. In this study, we exposed mice to 0 or 5.4 Gy TBI, collected urine samples periodically over 1 year, and assayed urine metabolites by using mass spectrometry. Longitudinal data analysis within the first year post-TBI revealed that cancers, including hematopoietic, solid, and benign neoplasms, could be distinguished by unique urinary signatures as early as 3 months post-TBI. Furthermore, a distinction among different types of malignancies could be clearly delineated as early as 3 months post-TBI for hematopoietic neoplasms, 6 months for solid neoplasms, and by 1 year for benign neoplasms. Moreover, the feature profile for radiation-exposed mice 6 months post-TBI was found to be similar to nonirradiated control mice at 18 months, suggesting that TBI accelerates aging. These results demonstrate that urine feature profiles following TBI can identify cancers in mice prior to macroscopic detection, with important implications for the early diagnosis and treatment. Cancer Res; 76(6); 1569–77. ©2016 AACR.

Cytotoxic therapies prime adaptive immune responses to cancer by stimulating the release of tumor-associated antigens. However, the tumor microenvironment into which these antigens are released is typically immunosuppressed, blunting the ability to initiate immune responses. Recently, activation of the DNA sensor molecule STING by cyclic dinucleotides was shown to stimulate infection-related inflammatory pathways in tumors. In this study, we report that the inflammatory pathways activated by STING ligands generate a powerful adjuvant activity for enhancing adaptive immune responses to tumor antigens released by radiotherapy. In a murine model of pancreatic cancer, we showed that combining CT-guided radiotherapy with a novel ligand of murine and human STING could synergize to control local and distant tumors. Mechanistic investigations revealed T-cell–independent and TNFα-dependent hemorrhagic necrosis at early times, followed by later CD8 T-cell–dependent control of residual disease. Clinically, STING was found to be expressed extensively in human pancreatic tumor and stromal cells. Our findings suggest that this novel STING ligand could offer a potent adjuvant for leveraging radiotherapeutic management of pancreatic cancer. Cancer Res; 76(1); 50–61. ©2015 AACR.

GNAQ and GNA11 are heterotrimeric G protein alpha subunits, which are mutated in a mutually exclusive pattern in most cases of uveal melanoma, one of the most aggressive cancers. Here we introduce the first transgenic mouse model of uveal melanoma, which develops cancers induced by expression of oncogenic GNAQQ209L under control of the Rosa26 promoter. Disease penetrance is 100% by 3 months of age, with 94% of mice also developing lung tumors. In this model, the Yap protein of the Hippo pathway is activated in the eyes, and blood vessels near the lesions in the head and lungs exhibit melanocytic invasion. While full transcription levels are not necessary for GNAQQ209L to transform mouse melanocytes, we obtained suggestive evidence of a selective advantage for increased GNAQQ209L expression in human tumors. Intriguingly, enforced expression of GNAQQ209L progressively eliminated melanocytes from the interfollicular epidermis in adults, possibly explaining the near absence of GNAQQ209 mutations in human epithelial melanomas. The mouse model also exhibited dermal nevi and melanocytic neoplasms of the central nervous system, accompanied by impaired hearing and balance, identifying a novel role for GNAQ in melanocyte-like cells of the inner ear. Overall, this model offers a new tool to dissect signaling by oncogenic GNAQ and to test potential therapeutics in an in vivo setting where GNAQQ209L mutations contribute to both the initiation and metastatic progression of uveal melanoma. Cancer Res; 75(16); 3384–97. ©2015 AACR.

Nitric oxide synthases (NOS) are important mediators of progrowth signaling in tumor cells, as they regulate angiogenesis, immune response, and immune-mediated wound healing. Ionizing radiation (IR) is also an immune modulator and inducer of wound response. We hypothesized that radiation therapeutic efficacy could be improved by targeting NOS following tumor irradiation. Herein, we show enhanced radiation-induced (10 Gy) tumor growth delay in a syngeneic model (C3H) but not immunosuppressed (Nu/Nu) squamous cell carcinoma tumor-bearing mice treated post-IR with the constitutive NOS inhibitor NG-nitro-l-arginine methyl ester (L-NAME). These results suggest a requirement of T cells for improved radiation tumor response. In support of this observation, tumor irradiation induced a rapid increase in the immunosuppressive Th2 cytokine IL10, which was abated by post-IR administration of L-NAME. In vivo suppression of IL10 using an antisense IL10 morpholino also extended the tumor growth delay induced by radiation in a manner similar to L-NAME. Further examination of this mechanism in cultured Jurkat T cells revealed L-NAME suppression of IR-induced IL10 expression, which reaccumulated in the presence of exogenous NO donor. In addition to L-NAME, the guanylyl cyclase inhibitors ODQ and thrombospondin-1 also abated IR-induced IL10 expression in Jurkat T cells and ANA-1 macrophages, which further suggests that the immunosuppressive effects involve eNOS. Moreover, cytotoxic Th1 cytokines, including IL2, IL12p40, and IFNγ, as well as activated CD8+ T cells were elevated in tumors receiving post-IR L-NAME. Together, these results suggest that post-IR NOS inhibition improves radiation tumor response via Th1 immune polarization within the tumor microenvironment. Cancer Res; 75(14); 2788–99. ©2015 AACR.

Studies in animals and humans show that blockade of nerve growth factor (NGF) attenuates both malignant and nonmalignant skeletal pain. While reduction of pain is important, a largely unanswered question is what other benefits NGF blockade might confer in patients with bone cancer. Using a mouse graft model of bone sarcoma, we demonstrate that early treatment with an NGF antibody reduced tumor-induced bone destruction, delayed time to bone fracture, and increased the use of the tumor-bearing limb. Consistent with animal studies in osteoarthritis and head and neck cancer, early blockade of NGF reduced weight loss in mice with bone sarcoma. In terms of the extent and time course of pain relief, NGF blockade also reduced pain 40% to 70%, depending on the metric assessed. Importantly, this analgesic effect was maintained even in animals with late-stage disease. Our results suggest that NGF blockade immediately upon detection of tumor metastasis to bone may help preserve the integrity and use, delay the time to tumor-induced bone fracture, and maintain body weight. Cancer Res; 74(23); 7014–23. ©2014 AACR.

Radiotherapy can produce antitumor benefits beyond the local site of irradiation, an immune-based phenomenon known as the abscopal effect, but the mechanisms underlying these benefits are poorly understood. Preclinical studies of ECI301, a mutant derivative of macrophage inhibitory protein-1α, have shown that its administration can improve the antitumor effects of radiotherapy in a manner associated with a tumor-independent abscopal effect. In this article, we report that i.v. administration of ECI301 after intratumoral injection of tumor cell lysates can inhibit tumor growth, not only at the site of injection but also at nontreated sites. Effects of the tumor lysate were further recapitulated by i.v. administration of the alarmins HSP70 or HMGB1, but not HSP60, and combinations of ECI301 + HSP70 were sufficient to inhibit tumor growth. Although injection of ECI301 + HMGB1 did not inhibit tumor growth, we found that administration of a neutralizing HMGB1 antibody neutralized the cooperative effects of ECI301 on tumor irradiation. Moreover, mice genetically deficient in TLR4, an immune pattern receptor that binds alarmins, including HMGB1 and HSP70, did not exhibit antitumor responses to irradiation with ECI301 administration. Although ECI301 was cleared rapidly from peripheral blood, it was found to bind avidly to HSP70 and HMGB1 in vitro. Our results suggest a model in which sequential release of the alarmins HSP70 and HMGB1 from a tumor by irradiation may trap circulating ECI301, thereby licensing or restoring tumor immunosurveillance capabilities of natural killer cells or CD4+ and CD8+ T cells against tumor cells that may evade irradiation. Cancer Res; 74(18); 5070–8. ©2014 AACR.

Cancer in a host induces responses that increase the ability of the microenvironment to sustain the growing mass, for example, angiogenesis, but cancer cells can have varying sensitivities to these sustainability signals. Here, we show that these sensitivities are significant determinants of ultimate tumor fate, especially in response to treatments and immune interactions. We present a mathematical model of cancer–immune interactions that modifies generalized logistic growth with both immune-predation and immune-recruitment. The role of a growing environmental carrying capacity is discussed as a possible regulatory mechanism for tumor growth, and this regulation is shown to modify cancer–immune interactions and the possibility of achieving immune-induced tumor dormancy. This mathematical model qualitatively matches experimental observations of immune-induced tumor dormancy as it predicts dormancy as a transient period of growth that necessarily ends in either tumor elimination or tumor escape. As dormant tumors may exist asymptomatically and may be easier to treat with conventional therapy, an understanding of the mechanisms behind tumor dormancy may lead to new treatments aimed at prolonging the dormant state or converting an aggressive cancer to the dormant state. Cancer Res; 73(12); 3534–44. ©2013 AACR.

GITR is a T-cell costimulatory receptor that enhances cellular and humoral immunity. The agonist anti-mouse GITR antibody DTA-1 has demonstrated efficacy in murine models of cancer primarily by attenuation of Treg-mediated immune suppression, but the translatability to human GITR biology has not been fully explored. Here, we report the potential utility of MK-4166, a humanized GITR mAb selected to bind to an epitope analogous to the DTA-1 epitope, which enhances the proliferation of both naïve and tumor-infiltrating T lymphocytes (TIL). We also investigated the role of GITR agonism in human antitumor immune responses and report here the preclinical characterization and toxicity assessment of MK-4166, which is currently being evaluated in a phase I clinical study. Expression of human GITR was comparable with that of mouse GITR in tumor-infiltrating Tregs despite being drastically lower in other human TILs and in many human peripheral blood populations. MK-4166 decreased induction and suppressive effects of Tregs in vitro. In human TIL cultures, MK-4166 induced phosphorylation of NFκB and increased expression of dual specificity phosphatase 6 (DUSP6), indicating that MK-4166 activated downstream NFκB and Erk signaling pathways. Furthermore, MK-4166 downregulated FOXP3 mRNA in human tumor infiltrating Tregs, suggesting that, in addition to enhancing the activation of TILs, MK-4166 may attenuate the Treg-mediated suppressive tumor microenvironment. Cancer Res; 77(16); 4378–88. ©2017 AACR.

Microbubble contrast agents can specifically deliver nucleic acids to target tissues when exposed to ultrasound treatment parameters that mediate microbubble destruction. In this study, we evaluated whether microbubbles and ultrasound-targeted microbubble destruction (UTMD) could be used to enhance delivery of EGF receptor (EGFR)–directed siRNA to murine squamous cell carcinomas. Custom-designed microbubbles efficiently bound siRNA and mediated RNAse protection. UTMD-mediated delivery of microbubbles loaded with EGFR-directed siRNA to murine squamous carcinoma cells in vitro reduced EGFR expression and EGF-dependent growth, relative to delivery of control siRNA. Similarly, serial UTMD-mediated delivery of EGFR siRNA to squamous cell carcinoma in vivo decreased EGFR expression and increased tumor doubling time, relative to controls receiving EGFR siRNA-loaded microbubbles but not ultrasound or control siRNA-loaded microbubbles and UTMD. Taken together, our results offer a preclinical proof-of-concept for customized microbubbles and UTMD to deliver gene-targeted siRNA for cancer therapy. Cancer Res; 72(23); 6191–9. ©2012 AACR.

Understanding metastasis is integral to curative cancer treatments. Using a mouse genetic screening model, we identified Merm1/Wbscr22 as a novel metastasis promoter that includes a methyltransferase fold in its structure. Merm1 showed high levels of expression in invasive breast cancer. Ectopic expression of Merm1 in nonmetastatic cells enhanced metastasis formation without affecting cell growth and motility. The intact methyltransferase fold of Merm1 was required for metastasis formation. Interestingly, Merm1 expression promoted cell survival after entrapment in the lung microvasculature. Consistent with these results, knockdown of endogenous Merm1 in tumor cells reduced lung retention and metastasis formation. On the basis of comparative transcriptome analysis, Merm1 expression was negatively correlated with the expression of tumor suppressor Zac1. We confirmed that Merm1 suppressed Zac1 expression with histone H3 methylation at Lys9 in the Zac1 promoter region. Zac1 can induce apoptosis through its ability to transcriptionally coactivate p53, which regulates apoptosis in the vasculature and is often downregulated in metastasis. We found that Zac1 knockdown reduced the p53-dependent apoptosis that was enhanced by Merm1 knockdown, thereby increasing lung retention of metastatic cells. Our findings show that Merm1 enhances cancer cell survival in the vasculature by suppressing Zac1/p53-dependent apoptosis, thereby enhancing metastasis. Cancer Res; 71(3); 1146–55. ©2010 AACR.

Genetic background affects susceptibility to pancreatic ductal adenocarcinoma in the Ela-KRASG12D mouse model. In this model, KRAS oncogene expression is driven by an elastase promoter in acinar cells of the pancreas on an FVB/NTac (FVB) background [FVB-Tg(Ela-KRASG12D)] with the transgene carried on the Y chromosome. Through linkage analysis of crosses between the C57BL/6J (B6), BALB/cJ (BALB), and DBA/2J (D2) inbred strains of mice and resistant FVB-Tg(Ela-KRASG12D), we have identified six susceptibility loci that affect mean preinvasive lesion multiplicity. Markers on chromosome 2 segregated with high tumor multiplicity in all three strains; these loci were designated Prsq1-3 (pancreatic ras susceptibility quantitative trait loci 1-3; combined F2 and N2 LODW, 6.0, 4.1, and 2.7, respectively). Susceptibility loci on chromosome 4, designated Prsq4 and Prsq5, were identified in crosses between FVB transgenic mice and B6 or BALB mice (combined F2 and N2 LODW, 3.6 and 2.9, respectively). A marker on chromosome 12 segregated with tumor multiplicity in a BALB × FVB-Tg(Ela-KRASG12D) cross and was designated Prsq6 (LODW, ∼2.5). B6-Chr YFVB-Tg(Ela-KRASG12D) and BALB-Chr YFVB-Tg(Ela-KRASG12D) consomics, which carry the KRAS transgene on the FVB Y chromosome on an otherwise inbred B6 or BALB background, developed ∼4-fold (B6) and ∼10-fold (BALB) more lesions than FVB-Tg(Ela-KRASG12D) mice. By 12 months of age, 10% of BALB-Chr YFVB-Tg(Ela-KRASG12D) mice developed invasive carcinomas. Our findings provide evidence that regions of chromosomes 2, 4, and 12 influence the development and progression of pancreatic neoplasms initiated by an oncogenic allele of KRAS in mice. Cancer Res; 70(21); 8398–406. ©2010 AACR.

The transcription factor NF-E2-related factor 2 (Nrf2) binds the antioxidant DNA response element (ARE) to activate important cellular cytoprotective defense systems. Recently several types of cancers have been shown to overexpress Nrf2, but its role in the cellular response to radiation therapy has yet to be fully determined. In this study, we report that single doses of ionizing radiation from 2 to 8 Gy activate ARE-dependent transcription in breast cancer cells in a dose-dependent manner, but only after a delay of five days. Clinically relevant daily dose fractions of radiation also increased ARE-dependent transcription, but again only after five days. Downstream activation of Nrf2-ARE-dependent gene and protein markers, such as heme oxygenase-1, occurred, whereas Nrf2-deficient fibroblasts were incapable of these responses. Compared with wild-type fibroblasts, Nrf2-deficient fibroblasts had relatively high basal levels of reactive oxygen species that increased greatly five days after radiation exposure. Further, in vitro clonogenic survival assays and in vivo sublethal whole body irradiation tests showed that Nrf2 deletion increased radiation sensitivity, whereas Nrf2-inducing drugs did not increase radioresistance. Our results indicate that the Nrf2-ARE pathway is important to maintain resistance to irradiation, but that it operates as a second-tier antioxidant adaptive response system activated by radiation only under specific circumstances, including those that may be highly relevant to tumor response during standard clinical dose-fractionated radiation therapy. Cancer Res; 70(21); 8886–95. ©2010 AACR.

It is widely accepted that redox status, along with the partial pressure of oxygen (pO2), determines the efficacy of some therapeutic methods applied to treat tumors, including radiation. Redox status, evaluated by the reduction of a nitroxyl probe, was reportedly heterogeneous in a mouse tumor model. However, neither variation of heterogeneity of the redox status among mice nor the relation of the redox status to pO2 in tumors has been characterized sufficiently. In this study, the regional reduction status in a mouse radiation-induced fibrosarcoma tumor model was evaluated using sequential three-dimensional electron paramagnetic resonance (EPR) imaging after i.v. injection of a tissue-permeable nitroxyl probe, HM-PROXYL. The regional decay of HM-PROXYL signal obeyed first-order kinetics, and the amplitude of the reduction rate and extent of its heterogeneity in a tumor varied among six mice. The tissue pO2 was measured using EPR oximetry with lithium phthalocyanine (LiPc) microcrystals implanted within the tumor. The location of LiPc was determined with EPR imaging. A sequential image was obtained following the injection of HM-PROXYL, even after LiPc implantation, by choosing an HM-PROXYL signal peak which does not overlap with the signal of LiPc. The relationship between pO2 and the reduction rate at the region of pO2 measurement was found to be low (r = 0.357) in 13 tumor-bearing mice, indicating that the extent of oxygenation does not necessarily affect the redox status under air-breathing conditions. The results strongly indicate the necessity of measurements of both redox status and oxygenation in every tumor to characterize tumor physiology. Cancer Res; 70(10); 4133–40. ©2010 AACR.

Although Bacillus Calmette-Guerin (BCG) intravesical therapy is a standard treatment for bladder cancer, eventual failure of response is a major problem. Treatments that can augment BCG therapy are urgently needed. We investigated whether curcumin, a component of Curcuma longa (also called turmeric), has potential to improve the current therapy using in vitro and in vivo MBT-2 murine tumor models. We found that curcumin potentiated BCG-induced apoptosis of human bladder cancer cells. BCG stimulated the release of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) from peripheral mononuclear neutrophils in a dose- and time-dependent manner, whereas curcumin enhanced the upregulation of TRAIL receptors. Electrophoretic mobility shift assay revealed that curcumin also suppressed the BCG-induced activation of the cell survival transcription factor NF-κB. In a syngeneic bladder cancer model, curcumin alone reduced the bladder tumor volume, but a significantly greater reduction was observed when BCG and curcumin were used in combination (P < 0.0001 versus control; P < 0.003 versus BCG alone). This was accompanied by a significant decrease in the proliferation marker Ki-67 (P < 0.01 versus control; P < 0.01 versus BCG alone) and microvessel density (CD31; P < 0.01 versus control; P < 0.01 versus BCG alone), decreased NF-κB in tumor tissue compared with the control, induced apoptosis, and decreased cyclin D1, vascular endothelial growth factor, cyclooxygenase-2, c-myc, and Bcl-2 expression in the tumor tissue. Upregulation of TRAIL receptor by the combination was also observed in tumor tissues. Overall, our results suggest that curcumin potentiates the antitumor effect of BCG through the inhibition of NF-κB and induction of TRAIL receptors in bladder cancer cells. [Cancer Res 2009;69(23):8958–66]

Lung cancer is the leading cause of cancer death worldwide. Here, we describe a genome-wide association study of chemically induced lung tumorigenesis on 593 mice from 21 inbred strains using 115,904 genotyped and 1,952,918 imputed single nucleotide polymorphisms (SNPs). Using a genetic background–controlled genome search, we identified a novel lung tumor susceptibility gene Las2 (Lung adenoma susceptibility 2) on distal chromosome 18. Las2 showed strong association with resistance to tumor induction (rs30245983; P = 1.87 × 10−9) as well as epistatic interactions (P = 1.71 × 10−3) with the pulmonary adenoma susceptibility 1 locus, a major locus affecting mouse lung tumor development (rs13459098, P = 5.64 × 10−27). Sequencing analysis revealed four nonsynonymous SNPs and two insertions/deletions in the susceptible allele of Las2, resulting in the loss of tumor suppressor activities in both cell colony formation and nude mouse tumorigenicity assays. Deletion of LAS2 was observed in ∼40% of human lung adenocarcinomas, implying that loss of function of LAS2 may be a key step for lung tumorigenesis. [Cancer Res 2009;69(15):6290–8]

Nonmyeloablative allogeneic stem cell transplantation (SCT) has been used for various malignancies, although detailed mechanisms of antitumor effects remain unclear. We showed that a nonmyeloablative allogeneic SCT regimen, which consists of mixed chimerism induced by an injection of donor spleen and bone marrow cells followed by cyclophosphamide treatment and a donor lymphocyte infusion (DLI), exerted antitumor effects on established murine bladder tumor, MBT-2. An expansion of donor CD4 T cells accompanied by transient but vigorous IFN-γ production was detected shortly after DLI. In vivo neutralization of IFN-γ or depletion of CD4 T cells from DLI abolished the antitumor effects, indicating an indispensable role of donor CD4 T cells producing IFN-γ. Donor as well as host CD8 T cells accumulated in the tumor region with time. Importantly, depletion of CD8 T cells from DLI did not reverse the suppression of tumor growth, indicating that CD4 T cells play a more essential role in mediating early antitumor effects. Furthermore, tumor-specific response of host CD8 T cells was suggested. These results not only provide the first evidence of nonmyeloablative allogeneic SCT for the treatment of bladder tumor but also elucidate detailed mechanisms of antitumor effects provoked by DLI. [Cancer Res 2009;69(12):5151–8]

Inflammatory bowel disease, including ulcerative colitis and Crohn's disease, substantially increases the risk of colorectal cancer. However, mechanisms linking mucosal inflammation to the sequence of dysplasia are incompletely understood. Whereas studies have shown oxidative damage to the colon, this study tests whether genotoxicity is elicited systemically by acute and chronic intestinal inflammation. In this study, genotoxic endpoints were assessed in peripheral leukocytes (DNA single- and double-stranded breaks and oxidative DNA damage) and normochromatic erythrocytes (micronuclei) during chemical or immune-mediated colitis. During three consecutive cycles of intestinal inflammation induced by dextran sulfate sodium administration, genotoxicity to peripheral leukocytes and erythroblasts was detected in both acute and chronic phases of dextran sulfate sodium–induced inflammation. Reactive oxygen species–mediated oxidative stress and DNA damage was confirmed with positive 8-oxoguanine and nitrotyrosine staining in peripheral leukocytes. Levels of DNA damage generally decreased during remission and increased during treatment, correlating with clinical symptoms and systemic inflammatory cytokine levels. In Gαi2−/− and interleukin-10−/− transgenic mice susceptible to immune-mediated colitis and inflammation-associated adenocarcinoma, similar levels of peripheral leukocyte and erythroblast genotoxicity were also observed. Moreover, this systemic genotoxicity was observed in mice with subclinical inflammation, which was further elevated in those with severe mucosal inflammation. We propose that mucosal inflammation, by eliciting substantial and ongoing systemic DNA damage, contributes early on to genetic instability necessary for progression to inflammatory bowel disease–associated dysplasia and the development of cancer. [Cancer Res 2009;69(11):4827–34]

金沢大学がん研究所がん幹細胞研究センター, Mutations in bone morphogenetic protein (BMP) receptor IA (BMPRlA) are responsible for a subset of cases of juvenile polyposis(JF) syndrome that develops hamartomatous tumors in the gastrointestinal tract. Mouse genetic studies have shown thatsuppression of BMP signaling in the intestines causes JP-type hamartoma development. Here, we generated K19-Nog transgenic miceexpressing noggin, a BMP antagonist, in gastric epithelium. However, inhibition of BMP signaling did not cause gastric phenotypes.We thus crossed K19-Nog with K19-C2mE mice that expressed Ptgs2 and Ptges in the stomach to generate compound transgenic mice. Expression of Ptgs2 and Ptges results in prostaglandin E2 (PGE2) biosynthesis, and both enzymes are induced in most human gastrointestinal tumors. Importantly, K19-Nog/ C2mE compound mice developed gastric hamartomas that were morphologicallysimilar to those found in JP with mucin-containing dilated cysts and inflammatory infiltration. Notably, treatment of K19-Nog/C2mE mice with a cyclo-oxygenase-2 inhibitor, celecoxib, significantly reduced tumor size with suppression of angiogenesis, suggesting that induction of the FGE2 pathway together with inhibition of BMP signaling is required for gastric hamartoma development.Moreover, microarray analyses revealed that canonical Wnt signaling target genes were not induced in K19-Nog/ C2mE hamartomas, indicating that BMP inhibition and PGE2 induction lead to gastric hamartoma development indepen- dent of the Wnt/ß-catenin pathway. These results, taken together, suggest that the FGE2 pathway is an effective preventive target against BMP-suppressed gastric hamartomas, as well as for Wnt/ß-catenin-activated adenocarcinomas. ©2009 American Association for Cancer Research.

The lack of safe and efficient systemic gene delivery vectors has largely reduced the potential of gene therapy in the clinic. Previously, we have reported that polypropylenimine dendrimer PPIG3/DNA nanoparticles are capable of tumor transfection upon systemic administration in tumor-bearing mice. To be safely applicable in the clinic, it is crucial to investigate the colloidal stability of nanoparticles and to monitor the exact biodistribution of gene transfer in the whole body of the live subject. Our biophysical characterization shows that dendrimers, when complexed with DNA, are capable of forming spontaneously in solution a supramolecular assembly that possesses all the features required to diffuse in experimental tumors through the enhanced permeability and retention effect. We show that these nanoparticles are of sizes ranging from 33 to 286 nm depending on the DNA concentration, with a colloidal stable and well-organized fingerprint-like structure in which DNA molecules are condensed with an even periodicity of 2.8 nm. Whole-body nuclear imaging using small-animal nano-single-photon emission computed tomography/computer tomography scanner and the human Na/I symporter (NIS) as reporter gene shows unique and highly specific tumor targeting with no detection of gene transfer in any of the other tissues of tumor-bearing mice. Tumor-selective transgene expression was confirmed by quantitative reverse transcription-PCR at autopsy of scanned animals, whereas genomic PCR showed that the tumor sites are the predominant sites of nanoparticle accumulation. Considering that NIS imaging of transgene expression has been recently validated in humans, our data highlight the potential of these nanoparticles as a new formulation for cancer gene therapy. [Cancer Res 2009;69(6):2655–62]

Notch signaling is required for vascular development and tumor angiogenesis. Although inhibition of the Notch ligand Delta-like 4 can restrict tumor growth and disrupt neovasculature, the effect of inhibiting Notch receptor function on angiogenesis has yet to be defined. In this study, we generated a soluble form of the Notch1 receptor (Notch1 decoy) and assessed its effect on angiogenesis in vitro and in vivo. Notch1 decoy expression reduced signaling stimulated by the binding of three distinct Notch ligands to Notch1 and inhibited morphogenesis of endothelial cells overexpressing Notch4. Thus, Notch1 decoy functioned as an antagonist of ligand-dependent Notch signaling. In mice, Notch1 decoy also inhibited vascular endothelial growth factor–induced angiogenesis in skin, establishing a role for Notch receptor function in this process. We tested the effects of Notch1 decoy on tumor angiogenesis using two models: mouse mammary Mm5MT cells overexpressing fibroblast growth factor 4 (Mm5MT-FGF4) and NGP human neuroblastoma cells. Exogenously expressed FGF4 induced Notch ligand expression in Mm5MT cells and xenografts. Notch1 decoy expression did not affect tumorigenicity of Mm5MT-FGF4 cells in vitro but restricted Mm5MT-FGF4 xenograft growth in mice while markedly impairing neoangiogenesis. Similarly, Notch1 decoy expression did not affect NGP cells in vitro but disrupted vessels and decreased tumor viability in vivo. These results strongly suggest that Notch receptor signaling is required for tumor neoangiogenesis and provides a new target for tumor therapy. [Cancer Res 2008;68(12):4727–35]

This study describes the application of a unique strategy to identify breast cancer antigens [tumor-associated antigen (TAA)]. In a mouse model, the strategy led to the identification of growth factor receptor–bound protein 10 (Grb10) as a newly identified TAA. Grb10 is a signal transduction molecule associated with multiple transmembrane tyrosine kinase receptors. It was discovered by comparing microarrays of cellular breast cancer vaccines highly enriched for cells that induced breast cancer immunity in tumor-bearing mice with nonenriched vaccines. The vaccines were prepared by transferring a cDNA expression library derived from SB5b cells, a breast cancer cell line C3H/He origin (H-2k), into LM mouse fibroblasts (H-2k). As the transferred cDNA integrates spontaneously into the genome of the recipient cells, replicates as the cells divide, and is expressed, the vaccine could be prepared from microgram amounts of tumor tissue. Relatively few cells in the transduced cell population, however, incorporated cDNA fragments that included genes specifying TAA. (The vast majority specified normal cellular constituents.) A unique strategy was used, therefore, to enrich the vaccine for immunotherapeutic cells. Twenty genes were overrepresented in the enriched vaccines. One, the gene for Grb10, was ∼100-fold overrepresented. To determine if Grb10 in the enriched vaccine was partly responsible for its therapeutic benefits, the gene was transferred into the fibroblast cell line, which was then used as a vaccine. Mice with established breast cancer treated solely by immunization with the modified fibroblasts developed robust immunity to the breast cancer cells, which, in some instances, was sufficient to result in tumor rejection. [Cancer Res 2008;68(7):2463–70]

Toll-like receptors (TLR) activate multiple steps in inflammatory reactions in innate immune responses. They also activate signals that are critically involved in the initiation of adaptive immune responses. Many tumorigenic chemicals have been associated with endotoxin hypersensitivity mediated through TLR4. To determine the role of TLR4 in cutaneous skin carcinogenesis, we treated TLR4-deficient C3H/HeJ mice and the TLR4-normal C3H/HeN mice with the carcinogenic polyaromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA). TLR4-deficient C3H/HeJ mice developed more tumors relative to the TLR4-normal C3H/HeN mice. Both C3H/HeN and C3H/HeJ mice developed a T-cell–mediated immune response to topically applied DMBA. Interestingly, the cell-mediated immune response was mediated by IFN-γ in C3H/HeN mice and by interleukin (IL)-17 in C3H/HeJ mice. Moreover, C3H/HeN mice had elevated circulating levels of IFN-γ following topical application of DMBA, whereas IL-17 was elevated in C3H/HeJ mice. The results of this study indicate that TLR4 plays an important role in the prevention of DMBA skin tumorigenesis and that this is associated with differences in the T-cell subtype activated. Efforts to divert the cell-mediated immune response from one that is IL-17 mediated to one that is IFN-γ mediated may prove to be beneficial in the prevention of DMBA-induced cutaneous tumors. [Cancer Res 2008;68(2):615–22]

Hypoxia is a common feature of solid tumors and is associated with their malignant phenotype. The transcription factor hypoxia-inducible factor-1 (HIF-1) is a major regulator of adaptation to hypoxia and is implicated in the malignant progression of cancers. Here, we studied whether hypoxia and HIF-1 expression contribute to the development of bone metastases using a well-characterized animal model of bone metastasis in MDA-MB-231 human breast cancer cells. To study the role of hypoxia in bone metastases, we tested the effects of the fusion protein (TOP3), the oxygen-dependent degradation domain of HIF-1α fused with HIV-TAT, and procaspase-3. TOP3 selectively induced apoptosis in hypoxic tumor cells in vitro and significantly reduced bone metastases in vivo. We next examined the role of HIF-1 in bone metastases by establishing MDA-MB-231 cells overexpressing constitutively active or dominant-negative HIF-1α (MDA/CA-HIF or MDA/DN-HIF, respectively). Bone metastases of MDA/CA-HIF were significantly increased with elevated number of CD31-positive blood vessels. In contrast, bone metastases were significantly reduced in MDA/DN-HIF. Because the progression of osteolytic bone metastases is due in part to the imbalance between bone formation and bone resorption, we examined the effects of hypoxia and HIF-1 on the differentiation of osteoblasts and osteoclasts. Hypoxia and CA-HIF overexpression markedly inhibited osteoblastic differentiation, whereas hypoxia increased osteoclast-like cell formation. In conclusion, these results suggest that tumor-associated hypoxia and HIF-1 expression promote the progression of bone metastases in breast cancer. Our results also suggest that hypoxia and HIF-1 lead to the development of osteolytic bone metastases by suppressing osteoblast differentiation and promoting osteoclastogenesis. [Cancer Res 2007;67(9):4157–63]

In the BCR/ABL DA1-3b mouse model of acute myelogenous leukemia, dormant tumor cells may persist in the host in a state of equilibrium with the CD8+ CTL-mediated immune response by actively inhibiting T cells. Dormant tumor cells also show a progressive decrease of suppressor of cytokine signaling 1 (SOCS1) gene expression and a deregulation of the Janus-activated kinase/signal transducers and activators of transcription (JAK/STAT) pathway due to methylation of the SOCS1 gene. Dormant tumor cells were more resistant to apoptosis induced by specific CTLs, but resistance decreased when SOCS1 expression was restored via demethylation or gene transfer. AG490 JAK2 inhibitor decreased the resistance of dormant tumor cells to CTLs, but MG132 proteasome inhibitor was effective only in SOCS1-transfected cells. Thus, SOCS1 regulation of the JAK/STAT pathways contributes to the resistance of tumor cells to CTL-mediated killing. Resistance of dormant tumor cells to apoptosis was also observed when induced by irradiation, cytarabine, or imatinib mesylate, but was reduced by SOCS1 gene transfer. This cross-resistance to apoptosis was induced by interleukin 3 (IL-3) overproduction by dormant tumor cells and was reversed with an anti–IL-3 antibody. Thus, tumor cells that remain dormant for long periods in the host in spite of a specific CTL immune response may deregulate their JAK/STAT pathways and develop cross-resistance to various treatments through an IL-3 autocrine loop. These data suggest possible new therapeutic targets to eradicate dormant tumor cells. [Cancer Res 2007;67(9):4491–8]

We have shown previously that endogenous deficiency of interleukin (IL)-12 promotes photocarcinogenesis in mice. To characterize the role of IL-12 deficiency in tumor angiogenesis, we developed IL-12p35 knockout (IL-12 KO) mice on a C3H/HeN background. IL-12 KO mice and their wild-type (WT) counterparts were subjected to a photocarcinogenesis protocol. When tumor yield was stabilized, samples of tumor and tumor-uninvolved UVB-exposed skin were collected and subjected to immunohistochemistry, gelatinolytic zymography, real-time PCR, and Western blot analysis of angiogenic factors. We found that the protein, mRNA expression and/or activity of the matrix metalloproteinases (MMP)-2, MMP-3, MMP-7, and MMP-9, and basic fibroblast growth factor, which play crucial roles in tumor growth, were significantly higher in UVB-exposed skin and tumors of IL-12 KO mice compared with WT mice. With respect to the tumor vasculature, the expression of CD31-positive cells and the expression of vascular endothelial growth factor were higher in the tumors of IL-12 KO mice than WTs. The proliferative capacity of tumor cells of the IL-12 KO mice was significantly higher than their WT counterparts when determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and by analyzing the expression of cyclin D1. The level of the proinflammatory cytokine IL-6 and the expression of IL-23 in tumors of IL-12 KO mice were markedly higher than in the tumors of WT mice. IL-23 has been shown to promote tumor growth. Together, these data indicate for the first time that IL-12 deficiency promotes proangiogenic stimuli in UVB-induced skin tumors and suggest that endogenous enhancement of IL-12 levels may be effective in the prevention and treatment of UV-induced skin cancers. [Cancer Res 2007;67(8):3785–93]

The central role of Src in the development of several malignancies, including breast cancer, and the accumulating evidence of its interaction with receptor tyrosine kinases, integrins, and steroid receptors have identified it as an attractive therapeutic target. In the current study, we have evaluated the effect of a Src/Abl kinase inhibitor, SKI-606, on breast cancer growth, migration, invasion, and metastasis. Treatment of human breast cancer cells MDA-MB-231 with SKI-606 caused a marked inhibition of cell proliferation, invasion, and migration by inhibiting mitogen-activated protein kinase and Akt phosphorylation. For in vivo studies, MDA-MB-231 cells transfected with the plasmid encoding green fluorescent protein (GFP; MDA-MB-231-GFP) were inoculated into the mammary fat pads of female BALB/c nu/nu mice. Once tumor volume reached 30 to 50 mm3, animals were randomized and treated with vehicle alone or 150 mg/kg SKI-606 by daily oral gavage. Experimental animals receiving SKI-606 developed tumors of significantly smaller volume (45–54%) compared with control animals receiving vehicle alone. Analysis of lungs, liver, and spleen of these animals showed a significant decrease in GFP-positive tumor metastasis in animals receiving SKI-606 at a dose that was well tolerated. Western blot analysis and immunohistochemical analysis of primary tumors showed that these effects were due to the ability of SKI-606 to block tumor cell proliferation, angiogenesis, growth factor expression, and inhibition of Src-mediated signaling pathways in vivo. Together, the results from these studies provide compelling evidence for the role of Src inhibitors as therapeutic agents for blocking breast cancer growth and metastasis. [Cancer Res 2007;67(4):1580–8]

Accumulation of tumor-associated macrophages (TAM) correlates with malignant progression, immune suppression, and poor prognosis. In this study, we defined a critical role for the cell-surface guidance molecule SEMA3A in differential proliferative control of TAMs. Tumor cell–derived SEMA3A restricted the proliferation of protumoral M2 macrophages but increased the proliferation of antitumoral M1, acting through the SEMA3A receptor neuropilin 1. Expansion of M1 macrophages in vivo enhanced the recruitment and activation of natural killer (NK) cells and cytotoxic CD8+ T cells to tumors, inhibiting their growth. In human breast cancer specimens, we found that immunohistochemical levels of SEMA3A correlated with the expression of genes characteristic of M1 macrophages, CD8+ T cells, and NK cells, while inversely correlating with established characters of malignancy. In summary, our results illuminate a mechanism whereby the TAM phenotype is controlled and identify the cell-surface molecule SEMA3A as a candidate for therapeutic targeting. Cancer Res; 76(11); 3166–78. ©2016 AACR.

Primary and metastatic bone cancers are difficult to eradicate and novel approaches are needed to improve treatment and extend life. As bone cancer grows, osteoclasts, the principal bone-resorbing cells of the body, are recruited to and activated at sites of cancer. In this investigation, we determined if osteoclast lineage cells could function as a cell-based gene delivery system to bone cancers. We used the cytosine deaminase (CD) 5-fluorocytosine (5-FC) enzyme/prodrug system and studied bone marrow and bones from transgenic mice expressing a novel CD gene regulated by the osteoclast tartrate-resistant acid phosphatase (TRAP) gene promoter (Tg/NCD). DsRed2-labeled 2472 sarcoma cells were placed in Tg/NCD osteoclastogenic cultures and treated with 5-FC. 5-FC treatment resulted in profound bystander killing (90%; P < 0.05). The effect of 5-FC treatment on osteoclast lineage cells was most dramatic when administered at the beginning of the 7-day cultures, suggesting that mature osteoclasts are less sensitive to 5-FC. Evaluation of osteoclast-directed bystander killing in vivo revealed dramatic killing of bone cancer with only a modest effect on osteoclast number. Specifically, 5-FC treatment of tumor-bearing Tg/NCD mice or Tg/NCD bone marrow transplanted C3H mice (Tg/NCD-C3H) resulted in 92% and 44% reductions in tumor area, respectively (P < 0.05). Eight of ten 5-FC-treated Tg/NCD mice had complete bone tumor killing and five of six 5-FC-treated Tg/NCD-C3H mice had reduced tumor compared with controls. In addition, Tg/NCD osteoclasts were resistant to 5-FC treatment in vivo, a very important feature, as it identifies osteoclasts as an ideal CD gene delivery system. (Cancer Res 2006; 66(22): 10929-35)

Emerging preclinical studies support the concept of a transient “normalization” of tumor vasculature during the early stage of antiangiogenic treatment, with possible beneficial effects on associated radiotherapy or chemotherapy. One key issue in this area of research is to determine whether this feature is common to all antiangiogenic drugs and whether the phenomenon occurs in all types of tumors. In the present study, we characterized the evolution of the tumor oxygenation (in transplantable liver tumor and FSAII tumor models) after administration of SU5416, an antagonist of the vascular endothelial growth factor receptor. SU5416 induced an early increase in tumor oxygenation [measured by electronic paramagnetic resonance (EPR)], which did not correlate with remodeling of the tumor vasculature (assessed by CD31 labeling using immunohistochemistry) or with tumor perfusion (measured by dynamic contrast enhanced-magnetic resonance imaging). Inhibition of mitochondrial respiration (measured by EPR) was responsible for this early reoxygenation. Consistent with these unique findings in the tumor microenvironment, we found that SU5416 potentiated tumor response to radiotherapy but not to chemotherapy. In addition to the fact that the characterization of the tumor oxygenation is essential to enable correct application of combined therapies, our results show that the long-term inhibition of oxygen consumption is a potential novel target in this class of compounds. (Cancer Res 2006; 66(19): 9698-704)

The most active metabolite of vitamin D, calcitriol, is growth inhibitory for various tumor types in vitro and in vivo and inhibits the growth of endothelial cells freshly isolated from tumors [tumor-derived endothelial cells (TDEC)]. We compared the effects of calcitriol on Matrigel-derived endothelial cells (MDEC) and TDEC isolated from Matrigel plugs and squamous cell carcinoma tumors, respectively. TDEC and MDEC expressed vitamin D receptor (VDR) and responded to calcitriol by increasing VDR protein expression. Although no mutations were found in VDR from either cell type, Scatchard plot analysis revealed a higher ligand-binding affinity in TDEC (Kd, 0.26 nmol/L) than MDEC (Kd, 0.65 nmol/L). The VDR signaling axis in both cells was intact as shown using nuclear translocation and 24-hydroxylase promoter-luciferase reporter assays. However, unlike TDEC, MDEC were resistant to calcitriol-induced growth inhibition. Calcitriol (10 nmol/L) resulted in a 12.3% growth inhibition of MDEC compared with 47% in TDEC. In TDEC, calcitriol resulted in induction of G0/G1 arrest (10.75%) and reduction of S-phase cells (6.8%) with induction of p27 and down-regulation of p21 protein expression. Apoptotic effects, determined by Annexin V staining were also observed in calcitriol-treated TDEC (38.6%). Calcitriol caused reduced expression of p-Erk and p-Akt and an increase of poly(ADP-ribose) polymerase and caspase-3 cleavage in TDEC. By contrast, none of these effects on cell cycle or apoptosis were seen in calcitriol-treated MDEC. These results show that TDEC were more sensitive than MDEC to the antiproliferative effects of calcitriol despite apparently normal VDR content and structure of signaling axis in both cell types. (Cancer Res 2006; 66(17): 8565-73)

Signal transducer and activator of transcription 1 (Stat1) is considered a key transcription factor that inhibits tumorigenesis, and Stat1 activation in the host is required for interleukin-12 (IL-12)–mediated generation of CTL activity. Using syngeneic Stat1−/− C3H mice bearing SCCVII tumors in this study, we discovered opposite results. Stat1 deficiency in the host significantly enhances IL-12–mediated tumor regression, resulting in tumor eradication from 60% of SCCVII tumor–bearing mice and significant inhibition of tumor growth when compared with control treatment (P < 0.01). This effect is independent of both Stat1-activating cytokine IFN-γ and Stat1-downstream effector molecule FasL because neither neutralization of IFN-γ nor knocking out of FasL enhances or inhibits IL-12–mediated tumor regression. IL-12 induces a high intensity of tumor-specific CTL activity in Stat1-deficient mice (P < 0.01), increases the CD8 T-cell density in tumor bearing Stat1−/− mice, and induces a T-cell–dependent tumor regression. The increased CTL activity and the high-intensity infiltration of T cells into the tumors in IL-12–treated Stat1−/− mice are likely due to the longer survival than the same cells from wild-type mice. Together, the data show that inhibition of Stat1 expression in the host enhances tumor-local IL-12 gene therapy for regressing tumors. This conclusion provides a new concept for designing an effective treatment strategy. (Cancer Res 2006; 66(8): 4461-7)

Up to 30% of patients with acute myeloid leukemia (AML) harbor internal tandem duplications (ITD) within the FLT3 gene, encoding a receptor tyrosine kinase. These mutations induce constitutive tyrosine kinase activity in the absence of the natural Flt3 ligand and confer growth factor independence, increased proliferation, and survival to myeloid precursor cells. The signaling pathways and downstream nuclear targets mediating leukemic transformation are only partly identified. Here, we show that the presence of Flt3-ITD constitutively activates Akt (PKB), a key serine-threonine kinase within the phosphatidylinositol 3-kinase pathway. Constitutive activation of Akt phosphorylated and inhibited the transcription factor Foxo3a. Restored Foxo3a activity reversed Flt3-ITD–mediated growth properties and dominant-negative Akt prevented Flt3-ITD–mediated cytokine independence. Conditional Akt activation targeted to the cell membrane induced cytokine-independent survival, cell cycle progression, and proliferation. Importantly, Akt activation was sufficient to cause in vitro transformation of 32D myeloid progenitor cells and in vivo promoted the development of a leukemia-like myeloid disease. Akt phosphorylation was found in myeloid blasts of 86% of AML patients, suggesting an important role in leukemogenesis. In summary, Akt is necessary for increased survival, proliferation, and leukemic transformation by Flt3-ITD, possibly by inactivation of Foxo transcription factors. These findings indicate that Akt and Foxo transcription factors are attractive targets for therapeutic intervention in AML.

We hypothesized that nonsteroidal antiinflammatory drugs (NSAIDs) might enhance tumor radiosensitivity by increasing tumor oxygenation (pO2), via either a decrease in the recruitment of macrophages or from inhibition of mitochondrial respiration. The effect of four NSAIDs (diclofenac, indomethacin, piroxicam, and NS-398) on pO2 was studied in murine TLT liver tumors and FSaII fibrosarcomas. At the time of maximum pO2 (tmax, 30 minutes after administration), perfusion, oxygen consumption, and radiation sensitivity were studied. Local pO2 measurements were done using electron paramagnetic resonance. Tumor perfusion and permeability measurements were assessed by dynamic contrast-enhanced magnetic resonance imaging. The oxygen consumption rate of tumor cells after in vivo NSAID administration was measured using high-frequency electron paramagnetic resonance. Tumor-infiltrating macrophage localization was done with immunohistochemistry using CD11b antibody. All the NSAIDs tested caused a rapid increase in pO2. At tmax, tumor perfusion decreased, indicating that the increase in pO2 was not caused by an increase in oxygen supply. Also at tmax, global oxygen consumption decreased but the amount of tumor-infiltrating macrophages remained unchanged. Our study strongly indicates that the oxygen effect caused by NSAIDs is primarily mediated by an effect on mitochondrial respiration. When irradiation (18 Gy) was applied at tmax, the tumor radiosensitivity was enhanced (regrowth delay increased by a factor of 1.7). These results show the potential utility of an acute administration of NSAIDs for radiosensitizing tumors, and shed new light on the mechanisms of NSAID radiosensitization. These results also provide a new rationale for the treatment schedule when combining NSAIDs and radiotherapy.

Expression of transporter associated with antigen processing (TAP) is often lost in metastatic carcinomas, resulting in defective antigen processing and presentation and escape of the cancer cells from immune surveillance. In this study, the nature of TAP deficiencies in tumors was investigated. By chromatin immunoprecipitation assay, we showed that the recruitment of RNA polymerase II to the TAP-1 gene was impaired in TAP-deficient cells derived from murine melanoma, prostate, and lung carcinomas, compared with TAP-expressing fibroblasts and lymphoma cells. This suggested that the deficiency in TAP-1 expression resulted, at least partially, from a relatively low level of transcription of the TAP-1 gene. Furthermore, levels of TAP-1 promoter activity, as assessed by stable transfections with a reporter construct containing the TAP-1 promoter, were relatively low in TAP-deficient cells. To examine genetic heritability of regulators of TAP-1 promoter activity, TAP- and MHC class I–deficient cells of H-2b origin were fused with wild-type fibroblasts of H-2k origin. Fusion with TAP-expressing cells complemented the low levels of TAP-1 promoter activity in TAP-deficient cells. However, these fused cells exhibited lower levels of TAP-1 mRNA and H-2k than unfused fibroblasts. Further analysis showed that TAP-1 mRNA stability was lower in fused carcinoma fibroblasts than in unfused fibroblasts. Based on these results, we propose that TAP deficiency in many carcinomas is caused by a decrease in activity/expression of trans-acting factors regulating TAP-1 promoter activity, as well as a decrease in TAP-1 mRNA stability. These results have significant implications for understanding immune evasion mechanisms in tumors.

Interleukin-8 (IL-8) is an angiogenic factor that promotes growth of pancreatic tumors. The purpose of this study was to determine if c-Src, a protein tyrosine kinase frequently overexpressed in pancreatic cancer, regulated IL-8 expression and to elucidate the Src-mediated signaling pathways that contribute to angiogenesis in pancreatic adenocarcinoma cells. In a panel of pancreatic cancer cell lines, expression of total and activated Src correlated with IL-8 production. Furthermore, ectopic expression of activated Src in PANC-1 cells with low endogenous Src activity significantly increased IL-8 production (P < 0.005). In contrast, pharmacologic inhibition of endogenous c-Src kinase activity or small interfering RNA–mediated “knockdown” of c-Src expression in L3.6pl cells with high Src expression and activity caused significant decreases in IL-8 production (P < 0.005). Inhibition of c-Src activity resulted in decreased phosphorylation of Akt, p38, and extracellular signal-regulated kinase (Erk)-1/2. Significant (P < 0.005) dose-dependent decreases were observed in IL-8 expression by inhibiting Src-dependent signaling molecules Erk-1/2 and p38 but not phosphatidylinositol 3-kinase. To assess the relevance of Src inhibition to angiogenesis, in vivo gelfoam assays were done. Robust infiltration of vessels was observed in gelfoam saturated with conditioned medium from pancreatic carcinoma cells. This angiogenesis was nearly abrogated in gelfoams saturated with conditioned medium from cells treated with the Src family kinase inhibitor, PP2 (P < 0.001). Thus, c-Src regulates critical “downstream” signaling pathways that contribute to expression of IL-8 in human pancreatic tumor cells, suggesting c-Src may be a target for therapeutic intervention in pancreatic adenocarcinoma.

Mutations of Apc and p53 have both been implicated in human and murine mammary neoplasia. To investigate potential interactions between Apc and p53, we conditionally inactivated Apc in both the presence and the absence of functional p53. Apc deficiency on its own leads to the development of metaplasia but not neoplasia. We show here that these areas of metaplasia are characterized by elevated levels of both p53 and p21. In the additional absence of p53,there is rapid progression to neoplasia, with 44.4% of lymphoma-free mice developing a mammary tumor with earliest observed onset at pregnancy. To investigate the mechanism by which p53 deficiency accelerates neoplasia, we used the Rosa26R reporter strain as a marker of Cre-mediated recombination and show a role for p53 in the loss of Apc-deficient cells. This role seems limited to pregnancy and subsequent time points. We therefore show clear synergy between these two mutations in mammary gland neoplasia and present data to suggest that at least one mechanism for this acceleration is the p53-dependent loss of Apc-deficient cells.

Darbepoietin alfa (DA) is a long-acting analogue of erythropoietin that has reduced receptor affinity and enhanced biological activity. Experiments were done to test the hypothesis that correction of anemia in tumor-bearing mice by DA would increase tumor oxygenation and potentiate radiation-induced tumor cell killing. A SCC VII tumor model was used to study tumor responses to fractionated radiation therapy in mice with anemia induced by total body irradiation. Administration of DA reduced the extent and duration of anemia and associated tumor hypoxia, protected the bone marrow cells and prevented the body weight loss from the effect of irradiation, and facilitated the recovery in a time-dependent manner, with the administration of DA prior to total body irradiation having the greatest protective effect. When combined with fractionated radiation therapy, DA increased the tumor growth delay time from 2.7 days for irradiation alone to 7.3 to 10.6 days for combination of DA and irradiation. The effect of DA on tumor responses to fractionated radiation therapy was observed when DA was given 18 to 4 days before starting radiation therapy, but DA was also equally effective as a radiosensitizer when given only 2 hours before fractionated irradiation therapy. Weekly dosing of DA was as efficacious for the enhancement of radiation responses of tumors as biweekly dosing. Similar results were obtained in the RIF-1 fibrosarcoma tumor model. These studies show that DA can effectively correct anemia in tumor-bearing mice and sensitize tumor cells to fractionated radiation therapy. Importantly, DA was also able to sensitize tumors to radiation in mice with uncorrected anemia and hypoxia, suggesting that the effect of DA on radiosensitivity was independent of these factors and a different mechanism of action may be responsible for this effect.

Hepatocellular carcinoma (HCC) is known to progress through a step often called tumor promotion. Phenobarbital (PB) is the prototype of nongenotoxic cacinogens that promote HCC in rodents. The molecular target of PB to elicit the promotion has been the subject of intense investigations over the last 30 years since it was discovered. The nuclear receptor constitutive active/androstane receptor (CAR) is activated by PB as well as by various other xenobiotics such as therapeutic drugs and environmental pollutants. CAR activation results in the transcriptional induction of numerous hepatic genes including those that encode xenobiotic-metabolizing enzymes such as a set of cytochrome P450s. In addition to PB, many CAR activators are nongenotoxic carcinogens, but the role of CAR in liver tumor promotion remains unexplored. Using Car−/− mice, we have here examined tumor promotion by chronic treatment with PB in drinking water after tumor initiation with a single dose of the genotoxic carcinogen diethylnitrosamine. None of the Car−/− mice developed either eosinophilic foci or advanced liver tumors, whereas all Car+/+ mice developed HCC and/or adenoma by 39 weeks. The results indicate that CAR is the molecular target of promotion by PB and that activation of this receptor is an essential requirement for liver tumor development.

The purpose of the present study was to evaluate both in vitro and in vivo a series of boron-containing nucleosides that potentially could be used as delivery agents for neutron capture therapy. The rationale for their synthesis was based on the fact that proliferating neoplastic cells have increased requirements for nucleic acid precursors, and, therefore, they should preferentially localize in the tumor. A series of 3-carboranlyalkyl thymidine analogs has been synthesized and a subset, designated N4, N5, and N7, and the corresponding 3-dihydroxypropyl derivatives, designated N4–2OH, N5–2OH, and N7–2OH, have been selected for evaluation. Using these compounds as substrates for recombinant human thymidine kinase-1 and the mitochondrial isoenzyme thymidine kinase-2, the highest phosphorylation levels relative to thymidine were seen with N5 and the corresponding dihydroxypropyl analog N5–2OH. In contrast, N4, N4-OH, N7, and N7-OH had substantially lower phosphorylation levels. To compare compounds with high and low thymidine kinase-1 substrate activity, N5 and N7 and the corresponding dihydroxypropyl derivatives were selected for evaluation of their cellular toxicity, uptake and retention by the F98 rat glioma, human MRA melanoma, and murine L929 cell lines, all of which are thymidine kinase-1(+), and a mutant L929 cell line that is thymidine kinase-1(−). N5–2OH was the least toxic (IC50, 43–70 μm), and N7 and N7–2OH were the most toxic (IC50, 18–49 μm). The highest boron uptake was seen with N7–2OH by the MRA 27 melanoma and L929 wild-type (wt) cell lines. The highest retention was seen with L929 (wt) cells, and this ranged from 29% for N5–2OH to 46% for N7. Based on the in vitro toxicity and uptake data, N5–2OH was selected for in vivo biodistribution studies either in rats bearing intracerebral implants of the F98 glioma or in mice bearing either s.c. or intracerebral implants of L929 (wt) tumors. At 2.5 hours after convection-enhanced delivery, the boron values for the F98 glioma and normal brain were 16.2 ± 2.3 and 2.2 μg/g, respectively, and the tumor to brain ratio was 8.5. Boron values at 4 hours after convection-enhanced delivery of N5–2OH to mice bearing intracerebral implants of L929 (wt) or L929 thymidine kinase-1(−) tumors were 39.8 ± 10.8 and 12.4 ± 1.6 μg/g, respectively, and the corresponding normal brain values were 4.4 and 1.6 μg/g, thereby indicating that there was selective retention by the thymidine kinase-1(+) tumors. Based on these favorable in vitro and in vivo data, neutron capture therapy studies will be initiated using N5–2OH in combination with two non-cell cycle dependent boron delivery agents, boronophenylalanine and sodium borocaptate.

Although clinical trials of antiangiogenic strategies have been disappointing when administered as single agents, such approaches can play an important role in cancer treatment when combined with conventional therapies. Previous studies have shown that DC101, an antiangiogenic monoclonal antibody against vascular endothelial growth factor receptor-2, can produce significant growth inhibition in spontaneous and transplanted tumors but can also induce substantial hypoxia. Because DC101 appears to potentiate radiotherapy in some tumors, the present studies were undertaken to characterize pathophysiological changes following combined therapy and to determine whether radioresponse is enhanced despite the induction of hypoxia. MCa-4 and MCa-35 mammary carcinomas were treated with: (a) DC101; (b) 5 × 6 Gy radiation fractions; or (c) the combination. Image analysis of frozen tumor sections was used to quantitate: (a) hypoxia; (b) spacing of total and perfused blood vessels; and (c) endothelial and tumor cell apoptosis. For MCa-4, combination treatment schedules produced significant and prolonged delays in tumor growth, whereas single-modality treatments had minor effects. For MCa-35, radiation or the combination led to equivalent growth inhibition. In all tumors, hypoxia increased markedly after either radiation or DC101 alone. Although combination therapy produced no immediate pathophysiological changes, hypoxia ultimately increased after cessation of therapy. Preferential increases in endothelial apoptosis following combination treatment suggest that in addition to blocking tumor angiogenesis, DC101 enhances radiotherapy by specifically sensitizing endothelial cells, leading to degeneration of newly formed blood vessels.

Tumor hypoxia has been reported to be a negative prognostic factor in a number of tumor sites. Both clinical and experimental studies have suggested a positive correlation between tumor hypoxia and increased metastatic efficiency; however, the mechanisms are not understood. In this study, the mechanisms of hypoxia-enhanced metastasis have been investigated in murine KHT fibrosarcoma and SCC VII cells. We have observed that hypoxia-pretreated KHT-C cells have a higher survival rate than control KHT-C cells after being arrested in mouse lungs. cDNA microarray analysis revealed many hypoxia-regulated genes, most of which have been reported to be involved in cell survival and growth. Among these genes, we have confirmed the up-regulation of Mdm2 by hypoxia and have demonstrated that this up-regulation is p53 independent. The up-regulation of Mdm2 by hypoxia is associated with decreased p53 protein and inhibition of the transactivation of p53 downstream proapoptotic genes. Overexpression of Mdm2 or suppression of p53 by transient transfection increased metastatic efficiency in KHT-C cells. These data suggest that hypoxia can increase tumor cell metastatic efficiency by rendering the tumor cells less sensitive to stress-induced cell death, e.g., through modifying the levels of Mdm2 and p53.

The TGFβ growth factor family member BMP4 is a potent suppressor of breast cancer metastasis. In the mouse, the development of highly metastatic mammary tumors is associated with an accumulation of myeloid-derived suppressor cells (MDSC), the numbers of which are reduced by exogenous BMP4 expression. MDSCs are undetectable in naïve mice but can be induced by treatment with granulocyte colony-stimulating factor (G-CSF/Csf3) or by secretion of G-CSF from the tumor. Both tumor-induced and G-CSF–induced MDSCs effectively suppress T-cell activation and proliferation, leading to metastatic enhancement. BMP4 reduces the expression and secretion of G-CSF by inhibiting NF-κB (Nfkb1) activity in human and mouse tumor lines. Because MDSCs correlate with poor prognosis in patients with breast cancer, therapies based on activation of BMP4 signaling may offer a novel treatment strategy for breast cancer. Cancer Res; 74(18); 5091–102. ©2014 AACR.

Biomarker studies have shown that expression of the T-cell coregulatory ligand PDL1 on tumor cells correlates with clinical responsiveness to the PD1 antibody nivolumab. Here, we report the findings of a preclinical cancer vaccine study demonstrating vaccine-dependent PDL1 upregulation in the tumor microenvironment. We formulated an IFNγ-inducing cancer vaccine called TEGVAX that combined GM-CSF and multiple Toll-like receptor agonists to increase the number of activated dendritic cells. Treatment of established tumors with TEGVAX retarded tumor growth in a manner associated with enhanced systemic antitumor immunity. Unexpectedly, TEGVAX also upregulated PDL1 expression in the tumor microenvironment, possibly explaining why tumors were not eliminated completely. In support of this likelihood, PDL1 upregulation in this setting relied upon IFNγ-expressing tumor-infiltrating CD4+ and CD8+ T cells and administration of a PD1-blocking antibody with TEGVAX elicited complete regression of established tumors. Taken together, our findings provide a mechanistic rationale to combine IFNγ-inducing cancer vaccines with immune checkpoint blockade. Cancer Res; 74(15); 4042–52. ©2014 AACR.

In an effort to develop cancer therapies that maximize cytotoxicity, while minimizing unwanted side effects, we studied a series of novel compounds based on the highly energetic heterocyclic scaffold, dinitroazetidine. In this study, we report the preclinical validation of 1-bromoacetyl-3,3-dinitroazetidine (ABDNAZ), a representative lead compound currently in a phase I clinical trial in patients with cancer. In tumor cell culture, ABDNAZ generated reactive free radicals in a concentration- and time-dependent manner, modulating intracellular redox status and triggering apoptosis. When administered to mice as a single agent, ABDNAZ exhibited greater cytotoxicity than cisplatin or tirapazamine under hypoxic conditions. However, compared with cisplatin, ABDNAZ was better tolerated at submaximal doses, yielding significant tumor growth inhibition in the absence of systemic toxicity. Similarly, when combined with radiation, ABDNAZ accentuated antitumor efficacy along with the therapeutic index. Toxicity studies indicated that ABDNAZ was not myelosuppressive and no dose-limiting toxicity was apparent following daily administration for 14 days. Taken together, our findings offer preclinical proof-of-concept for ABDNAZ as a promising new anticancer agent with a favorable toxicity profile, either as a chemotherapeutic agent or a radiosensitizer. Cancer Res; 72(10); 2600–8. ©2012 AACR.