Anti-angiogenic therapy subsequent to adeno-associated-virus-mediated immunotherapy eradicates lymphomas that disseminate to the liver.
Liver cancer has a very poor prognosis and lacks effective therapy. We have previously demonstrated that intraportal injection of adeno-associated-viral (AAV) particles that express angiostatin lead to long-term expression of angiostatin capable of suppressing the outgrowth of EL-4 tumors in the liver. Here we combine AAV-mediated angiostatin therapy with immunotherapy by employing an AAV vector encoding the T-cell costimulator B7.1. Incubation of EL-4 cells with AAV-B7.1 viruses resulted in the rapid expression of B7.1 on the surface of 80% of EL-4 cells. Mice that were vaccinated with B7.1-engineered tumor cells rejected the tumor cells and resisted a secondary challenge with unmodified parental cells. Splenocytes from the vaccinated mice were highly cytotoxic towards parental EL-4 cells in vitro. However, the vaccinated mice failed to resist the challenge of a heavy burden of EL-4 cells. Intraportal injection of AAV particles that express angiostatin into mice that had been vaccinated 1 month earlier with B7.1-engineered tumor cells protected mice against the challenge of a heavy burden of EL-4 cells and eradicated tumors that had disseminated to the liver. The combinational therapy increased the survival rate of mice with advanced liver cancer. These encouraging results warrant investigation of the employment of anti-angiogenic therapy subsequent to cancer immunotherapy for targeting unresectable disseminated liver metastases.

title: Beta7 integrins contribute to skin graft rejection.
abstract: Because integrins alpha4beta7 and alphaEbeta7 contribute to epidermotropism of T-cells during skin inflammation, we sought to study their role in skin allograft rejection.



Wild-type (WT) (beta7+/+) and beta7 gene knockout (beta7-/-) C57BL/6 (H-2(b)) mice and SJL/J (H-2(s)) mice served as donors and recipients of allogeneic skin grafts. An anti-integrin beta7 subunit mAb (FIB504.64) was used to treat WT beta7+/+ C57BL/6 recipients of skin grafts from SJL/J mice.



WT C57BL/6 recipients acutely rejected skin from SJL/J mice in 13 days. In contrast, the survival of SJL/J skin on either beta7-/- gene knockout or WT C57BL/6 recipients treated with anti-beta7 subunit mAb, was prolonged by 6 to 7 additional days (P<0.01). The survival of skin allografts from either beta7-/- or beta7+/+ C57BL/6 mice received by SJL/J recipients was not prolonged (P >0.05).



Beta7 integrins contribute to skin graft rejection, in accord with their role in mediating the epidermotropism of T-cells during skin inflammation.

title: Intramuscular delivery of antiangiogenic genes suppresses secondary metastases after removal of primary tumors
abstract: The success of surgery to remove primary tumors can be compromised by the subsequent outgrowth of metastases. It is recognized that primary tumors secrete antiangiogenic factors that suppress the outgrowth of their daughter metastases. In accord we show here that surgical removal of primary EL-4 lymphomas led to a marked decrease in the levels of circulating angiostatin and endostatin, and promoted the growth of distant nodular tumors. Expression vectors encoding angiostatin and endostatin, formulated with poly-N-vinyl pyrrolidone (PVP), were injected into the tibialis and gastrocnemia muscles, leading to expression of angiostatin and endostatin in muscle fibers. High levels of biologically active exogenous proteins were secreted into the circulation. Intramuscular gene therapy with angiostatin and endostatin plasmids significantly inhibited tumor vascularity and induced tumor cell apoptosis, and thereby suppressed the growth of secondary subcutaneous and disseminated metastatic tumors in the lung and liver. Simultaneous intramuscular delivery of both angiostatin and endostatin plasmids significantly prolonged the survival of mice after removal of primary tumors. These results suggest that intramuscular gene transfer of angiostatin and endostatin might serve as a prophylactic cancer-prevention strategy to combat the recurrence of cancer after surgical resection of primary tumors.

title: Overexpression of von Hippel-Lindau tumor suppressor protein and antisense HIF-1α eradicates gliomas
abstract: The von Hippel–Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the α subunits of hypoxia-inducible-factors responsible for stimulating tumor angiogenesis and glycolysis, and targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to tumorigenesis and development of sporadic renal cell carcinomas and central nervous system hemangioblastomas. In the present study, we investigated whether engineered overexpression of pVHL in C6 glioma cells, which already express endogenous pVHL, would suppress the tumorigenicity of this particular tumor cell type. C6 cells overexpressing VHL displayed a reduced growth rate (70% inhibition) compared to the parental cell line when subcutaneously implanted in athymic (nu/nu) mice. Growth inhibition was associated with a 50% reduction in the number of tumor vessels and a 60% increase in tumor cell apoptosis, due in part to downregulation of HIF-1, VEGF, and the antiapoptotic factor Bcl-2, respectively. Gene transfer of VHL suppressed the growth of established C6 gliomas, and synergized with antisense HIF-1 to completely eradicate tumors. The data suggest that VHL gene therapy and/or agents that increase VHL expression could have utility in the treatment of gliomas, particularly when combined with agents that inhibit the expression or function of HIF-1.

title: Regression of solid tumors by engineered overexpression of von Hippel–Lindau tumor suppressor protein and antisense hypoxia-inducible factor-1α
abstract: The von Hippel–Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the α subunits of hypoxia-inducible factors (HIFs) responsible for stimulating tumor angiogenesis and glycolysis, targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to the development of sporadic renal cell carcinomas (RCCs). In the present study, we sought to determine whether engineered overexpression of pVHL in tumors other than RCC can inhibit tumor growth, either as a monotherapy, or in combination with antisense HIF-1α therapy. Intratumoral injection of subcutaneous EL-4 thymic lymphomas with an expression plasmid encoding pVHL resulted in the downregulation of HIF-1α and vascular endothelial growth factor (VEGF). There was a concomitant reduction in tumor angiogenesis and increased tumor cell apoptosis due in part to downregulation of Bcl-2 expression. VHL therapy resulted in the complete regression of small (0.1 cm diameter) tumors whereas, in contrast, large (0.4 cm diameter) EL-4 tumors were only slowed in their growth. Nevertheless, large tumors completely regressed in response to intratumoral injection of a combination of antisense HIF-1α and VHL plasmids. Combination therapy resulted in increased losses of HIF-1α, VEGF, and tumor blood vessels, and increased tumor cell apoptosis. These novel results suggest that synergistic therapies that simultaneously block the expression or function of HIF-1α, and enhance the expression or function of VHL may be beneficial in the treatment of cancer.

title: Antisense HIF-1α prevents acquired tumor resistance to angiostatin gene therapy
abstract: Angiostatin is a naturally occurring inhibitor of angiogenesis that is being developed as a drug to fight cancer. In this study we reveal that EL-4 tumors established in mice rapidly develop resistance to angiostatin gene therapy by upregulating hypoxia-inducible pathways. Angiostatin initially delayed tumor growth for 6 days by reducing blood vessel density. However, tumors quickly responded by upregulating the production of hypoxia-inducible factor-1α (HIF-1α) and its effector vascular endothelial growth factor (VEGF) in response to increasing tumor hypoxia, leading to restored angiogenesis and rapid tumor growth. Theoretically, blockade of HIF-1 should prevent resistance to anti-angiogenic therapy by preventing a tumor from responding to induced hypoxia. Antisense HIF-1α inhibited the expression of HIF-1α and of the HIF-1 effectors VEGF, glucose transporter-1 and lactate dehydrogenase. As a monotherapy, it was effective in eradicating small 0.1 cm diameter tumors, but only delayed the growth of large 0.4 cm diameter tumors. In contrast, timed injection of a combination of angiostatin and antisense HIF-1α plasmids completely eradicated large EL-4 tumors within 2 weeks, and prevented upregulation of hypoxia-inducible pathways induced by angiostatin. The data indicate that blocking hypoxia-inducible pathways by antisense HIF-1α can circumvent hypoxia-induced drug resistance and thereby augment the efficacy of anti-angiogenic therapies.

title: Interruption of nuclear localization of ATBF1 during the histopathologic progression of head and neck squamous cell carcinoma.
abstract: The AT-motif binding factor 1 (ATBF1) gene is frequently altered at the genetic level in several types of cancer, but its protein expression and subcellular localization have not been well studied in human cancers, including head and neck squamous cell carcinomas (HNSCCs).



ATBF1 expression and localization were examined in 5 cell lines and 197 clinical specimens of HNSCC, and correlated with pathologic and clinical characteristics.



ATBF1 was predominantly localized in the nucleus of hyperplastic squamous epithelium. Whereas nuclear ATBF1 dramatically decreased in invasive tumors (p = .0012), cytoplasmic ATBF1 levels progressively increased from dysplasia to invasive tumors (p < .0001), and the increase correlated with poor survival. Reduced nuclear ATBF1 level was also detected in HNSCC cell lines.



Nuclear localization of ATBF1 is frequently interrupted in HNSCC, and the interruption is significantly associated with the progression of HNSCC. The cytoplasmic ATBF1 level could be useful for predicting patient survival.

title: Kruppel-like factor 5 (KLF5) is critical for conferring uterine receptivity to implantation
abstract: A blastocyst will implant only when the uterus becomes receptive. Following attachment, luminal epithelial cells undergo degeneration at the site of the blastocyst. Although many genes critical for uterine receptivity are primarily regulated by ovarian hormones, Kruppel-like factor 5 (KLF5), a zinc finger-containing transcription factor, is persistently expressed in epithelial cells independently of ovarian hormones. Loss of uterine Klf5 causes female infertility due to defective implantation. Cox2 is normally expressed in the luminal epithelium and stroma at the site of blastocyst attachment, but luminal epithelial COX2 expression is absent with loss of Klf5. This is associated with the retention of the epithelium around the implantation chamber with arrested embryonic growth. These results suggest that Klf5 is indispensable for normal implantation.

title: Tuning exchange bias in core/shell FeO/Fe3O4 nanoparticles.
abstract: Monodisperse 35 nm FeO nanoparticles (NPs) were synthesized and oxidized in a dry air atmosphere into core/shell FeO/Fe(3)O(4) NPs with both FeO core and Fe(3)O(4) shell dimensions controlled by reaction temperature and time. Temperature-dependent magnetic properties were studied on FeO/Fe(3)O(4) NPs obtained from the FeO NPs oxidized at 60 and 100 °C for 30 min. A large exchange bias (shift in the hysteresis loop) was observed in these core/shell NPs. The relative dimensions of the core and shell determine not only the coercivity and exchange field but also the dominant reversal mechanism of the ferrimagnetic Fe(3)O(4) component. This is the first time demonstration of tuning exchange bias and of controlling asymmetric magnetization reversal in FeO/Fe(3)O(4) NPs with antiferromagnetic core and ferrimagnetic shell.

title: In pursuit of leucine-rich repeat-containing G protein-coupled receptor-5 regulation and function in the uterus.
abstract: Leucine-rich repeat-containing G protein-coupled receptor (LGR)-5 is a recently identified marker of stem cells in adult intestinal epithelium and hair follicles. Because of this characteristic, we studied the status of Lgr5 expression in the mouse uterus under various conditions. Lgr5 is highly expressed in the uterine epithelium of immature mice and is dramatically down-regulated after the mice resume estrous cycles. Surprisingly, whereas its expression is up-regulated in uteri of ovariectomized mice, the expression is down-regulated by estrogen and progesterone via their cognate nuclear receptors, estrogen receptor-alpha and progesterone receptor, respectively. Using a mouse endometrial cancer model, we also found that Lgr5 is highly expressed in the epithelium during the initial stages of tumorigenesis but is remarkably down-regulated in fully developed tumors. Lgr5 is a downstream target of Wnt signaling in the intestine. Genetic evidence shows that either excessive or absence of Wnt signaling dampens Lgr5 expression in the uterus. Collectively, our results show that Lgr5 expression in the mouse uterine epithelium is unique and dynamically regulated under various physiological and pathological states of the uterus, suggesting that this orphan receptor has important functions in uterine biology. However, identifying definitive uterine function of LGR5 will require further investigation using conditional deletion of uterine Lgr5 because systemic deletion of this gene is neonatally lethal.

title: Characterization of Nuclear Localization and SUMOylation of the ATBF1 Transcription Factor in Epithelial Cells
abstract: ATBF1/ZFHX3 is a large transcription factor that functions in development, tumorigenesis and other biological processes. ATBF1 is normally localized in the nucleus, but is often mislocalized in the cytoplasm in cancer cells. The mechanism underlying the mislocalization of ATBF1 is unknown. In this study, we analyzed the nuclear localization of ATBF1, and found that ectopically expressed ATBF1 formed nuclear body (NB)-like dots in the nucleus, some of which indeed physically associated with promyelocytic leukemia (PML) NBs. We also defined a 3-amino acid motif, KRK2615-2617, as the nuclear localization signal (NLS) for ATBF1. Interestingly, diffusely distributed nuclear SUMO1 proteins were sequestered into ATBF1 dots, which could be related to ATBF1's physical association with PML NBs, known SUMOylation hotspots. Furthermore, ATBF1 itself was SUMOylated. ATBF1 SUMOylation occurred at more than 3 lysine residues including K2349, K2806 and K3258 and was nuclear specific. Finally, the PIAS3 SUMO1 E3 ligase, which interacts with ATBF1 directly, diminished rather than enhanced ATBF1 SUMOylation, preventing the co-localization of ATBF1 with SUMO1 in the nucleus. These findings suggest that nuclear localization and SUMOylation are important for the transcription factor function of ATBF1, and that ATBF1 could cooperate with PML NBs to regulate protein SUMOylation in different biological processes.