STAT3, HIF-1alpha, EPO and EPOR - signaling proteins in human primary ductal breast cancers

STAT3 upregulates expression of HIF-1 induced EPO. Receptor EPOR was reported to activate STAT3. Our study was aimed at demonstration of tissue immunoreactivities of those proteins and determination of their relationships in reference to clinicopathological variables of breast cancers. We detected STAT3, HIF-1alpha, EPO and EPOR in specimens of 76 human, female, ductal breast cancers by immunohistochemistry. STAT3 was detected in 38 of 76 cancers (50%). HIF-1alpha was found in 55 cases (72%). EPO positive tumors comprised 89% of all the cancers (68 cases). EPOR was also visualized in 55 cases (72%). Anti-HIF-1alpha and anti-STAT3 stained nuclei and cytoplasm of breast cancer cells in diffuse and finely granular fashion. Strong membranous expressions of EPO and EPOR were distributed in cytoplasmic and membranous granularity or diffuse staining. STAT3 correlated with HIF-1 in general (r=0.4012, p<0.0001) and in different patients' subgroups. STAT3 was significantly associated with EPO and EPOR in all the cancers (r=0.2370, p=0.039 and r=0.3336, p=0.003, respectively). Besides a correlation between STAT3 and EPOR in node negative ones, STAT3 wasn't related to EPO and EPOR in remaining subgroups. HIF-1alpha correlated with EPO and EPOR in most of analyzed groups. Immunoreactivity to EPO generally was associated with EPOR (r=0.3520, p=0.002). Statistically analyzed distributions of the proteins reflected functional dependences among STAT3, HIF-1alpha, EPO and EPOR in cellular signal conduction.     

2-Oxoglutarate downregulates expression of vascular endothelial growth factor and erythropoietin through decreasing hypoxia-inducible factor-1alpha and inhibits angiogenesis

In oxygenated cells, hypoxia-inducible factor-1 (HIF-1) alpha subunits are rapidly degraded by a mechanism that involves ubiquitination by the von Hippel-Lindau tumor suppressor E3 ligase complex using 2-oxoglutarate as a substrate. We examined the effect of 2-oxoglutarate on the production of erythropoietin and vascular endothelial growth factor (VEGF). The expression of erythropoietin and VEGF protein were dose-dependently downregulated in Hep3B cells by the addition of 2-oxoglutarate. The promoter activity of VEGF-luciferase was dose-dependently downregulated by the addition of 2-oxoglutarate. Gel mobility shift assays revealed that the addition of 2-oxoglutarate dose-dependently inhibited HIF-1 binding activity, but did not affect GATA binding activity. Western blot analysis revealed that 2-oxoglutarate dose-dependently inhibited the HIF-1alpha protein level in Hep3B cells in hypoxic conditions. However, MG132 (the proteasome inhibitor) rescued the inhibition of HIF-1alpha protein expression by 2-oxoglutarate. Furthermore, under hypoxic conditions, 2-oxoglutarate dose-dependently inhibited tube formation in in vitro angiogenesis assays. These results indicate that 2-oxoglutarate treatment may be useful for the inhibition of angiogenesis.     

Human CD4+ T cells present within the microenvironment of human lung tumors are mobilized by the local and sustained release of IL-12 to kill tumors in situ by indirect effects of IFN-gamma

By implanting nondisrupted pieces of human lung tumor biopsy tissues into SCID mice, it has been possible to establish viable grafts of the tumor, as well as the tumor-associated microenvironment, including inflammatory cells, fibroblasts, tumor vasculature, and the extracellular matrix. Using this xenograft model, we have evaluated and characterized the effects of a local and sustained release of human rIL-12 (rhIL-12) from biodegradable microspheres. In response to rhIL-12, the human CD45+ inflammatory cells present within the xenograft mediate the suppression or the complete arrest of tumor growth in SCID mice. Analysis of the cellular events reveals that human CD4+ and CD8+ T cells are induced by rhIL-12 to produce and secrete IFN-gamma. Serum levels of human IFN-gamma in mice bearing rhIL-12-treated tumor xenografts correlate directly with the degree of tumor suppression, while neutralizing Abs to human IFN-gamma abrogate the IL-12-mediated tumor suppression. Gene expression profiling of tumors responding to intratumoral rhIL-12 demonstrates an up-regulation of IFN-gamma and IFN-gamma-dependent genes not observed in control-treated tumors. Genes encoding a number of proinflammatory cytokines, chemokines (and their receptors), adhesion molecules, activation markers, and the inducible NO synthase are up-regulated following the introduction of rhIL-12, while genes associated with tumor growth, angiogenesis, and metastasis are decreased in expression. NO contributes to the tumor killing because an inhibitor of inducible NO synthase prevents IL-12-induced tumor suppression. Cell depletion studies reveal that the IL-12-induced tumor suppression, IFN-gamma production, and the associated changes in gene expression are all dependent upon CD4+ T cells.     

Near-infrared theranostic photoimmunotherapy (PIT): repeated exposure of light enhances the effect of immunoconjugate

Armed antibody-based targeted molecular therapies offer the possibility of effective tumor control with a minimum of side effects. Photoimmunotherapy (PIT) employs a monoclonal antibody-phototoxic phthalocyanine dye, IR700 conjugate, that is activated by focal near-infrared (NIR) light irradiation after antibody binding to the targeted tumor cell surface leading to rapid necrotic cell death. Therapy by single NIR light irradiation was effective without significant side effects; however, recurrences were seen in most treated mice probably because of inhomogeneous distribution of panitumumab-IR700 immunoconjugate in the tumor, leading to ineffective PIT. We describe here an optimized regimen of effective PIT method for the same HER1-overexpressing tumor model (A431) with fractionated administration of panitumumab-IR700 conjugate followed by systematic repeated NIR light irradiation to the tumor based on timing of antibody redistribution into the remnant tumor under the guidance of IR700 fluorescence signal. Eighty percent of the A431 tumors were eradicated with repeated PIT without apparent side effects and survived tumor-free for more than 120 days even after stopping therapy at day 30. Therapeutic effects were monitored using IR700 fluorescent signal. PIT is a promising highly selective and clinically feasible theranostic method for treatment of mAb-binding tumors with minimal off-target effects.     

Intrinsic and extrinsic erythropoietin enhances neuroprotection against ischemia and reperfusion injury in vitro

This study was designed to investigate the neuroprotective effect of intrinsic and extrinsic erythropoietin (EPO) against hypoxia/ischemia, and determine the optimal time-window with respect to the EPO-induced neuroprotection. Experiments were conducted using primary mixed neuronal/astrocytic cultures and neuron-rich cultures. Hypoxia (2%) induces hypoxia-inducible factor-1alpha (HIF-1alpha) activity followed by strong EPO expression in mixed cultures and weak expression in neuron-rich cultures as documented by both western blot and RT-PCR. Immunoreactive EPO was strongly detected in astrocytes, whereas EPOR was only detected in neurons. Neurons were significantly damaged in neuron-rich cultures but were distinctly rescued in mixed cultures. Application of recombinant human EPO (rhEPO) (0.1 U/mL) within 6 h before or after hypoxia significantly increased neuronal survival compared with no rhEPO treatment. Application of rhEPO after onset of reoxygenation achieved the maximal neuronal protection against ischemia/reperfusion injury (6 h hypoxia followed 24 h reoxygenation). Our results indicate that HIF-1alpha induces EPO gene released by astrocytes and acts as an essential mediator of neuroprotection, prove the protective role of intrinsic astrocytic-neuronal signaling pathway in hypoxic/ischemic injury and demonstrate an optimal therapeutic time-window of extrinsic rhEPO in ischemia/reperfusion injury in vitro. The results point to the potential beneficial effects of HIF-1alpha and EPO for the possible treatment of stroke.     

Beclin 1 deficiency is associated with increased hypoxia-induced angiogenesis

Beclin 1, a tumor suppressor protein, acts as an initiator of autophagy in mammals. Heterozygous disruption of Beclin 1 accelerates tumor growth, but the underlying mechanisms remain unclear. We examined the role of Beclin 1 in tumor proliferation and angiogenesis, using a primary mouse melanoma tumor model. Beclin 1 (Becn1 (+/-) ) hemizygous mice displayed an aggressive tumor growth phenotype with increased angiogenesis under hypoxia, associated with enhanced levels of circulating erythropoietin but not vascular endothelial growth factor, relative to wild-type mice. Using in vivo and ex vivo assays, we demonstrated increased angiogenic activity in Becn1 (+/-) mice relative to wild-type mice. Endothelial cells from Becn1 (+/-) mice displayed increased proliferation, migration and tube formation in response to hypoxia relative to wild-type cells. Moreover, Becn1 (+/-) cells subjected to hypoxia displayed increased hypoxia-inducible factor-2α (HIF-2α) expression relative to HIF-1α. Genetic interference of HIF-2α but not HIF-1α, dramatically reduced hypoxia-inducible proliferation, migration and tube formation in Becn1 (+/-) endothelial cells. We demonstrated that mice deficient in the autophagic protein Beclin 1 display a pro-angiogenic phenotype associated with the upregulation of HIF-2α and increased erythropoietin production. These results suggest a relationship between Beclin 1 and the regulation of angiogenesis, with implications in tumor growth and development.     

Bromodomain protein BRD4 promotes cell proliferation in skin squamous cell carcinoma

The present study examined the expression and biological functions of bromodomain-containing protein 4 (BRD4) in skin squamous cell carcinoma (SCC) cells. Our results show that BRD4 mRNA and protein expression was upregulated in human skin SCC cells, as compared to its level in the normal skin keratinocytes and fibroblasts. Treatment with BRD4 inhibitors, JQ1 and CPI203, resulted in proliferation inhibition, apoptosis and cell cycle arrest in both established (A431 cell line) and primary skin SCC cells. Furthermore, BRD4 knockdown (by targeted shRNAs) or knockout (by CRISPR/Cas9) largely inhibited A431 cell proliferation. Reversely, forced-overexpression of BRD4 in A431 cells facilitated cell proliferation. We show that BRD4 is required for the expression of several oncogenes, including cyclin D1, Bcl-2 and MYC. BRD4 inhibition, knockdown or knockout significantly decreased above oncogene expression in SCC cells. In vivo, CRISPR/Cas9-mediated BRD4 knockout significantly suppressed A431 xenograft tumor growth in severe combined immunodeficient (SCID) mice. Together, our results suggest that BRD4 could be a novel and pivotal oncogenic protein of skin SCC.     

Human CD4+ effector memory T cells persisting in the microenvironment of lung cancer xenografts are activated by local delivery of IL-12 to proliferate, produce IFN-gamma, and eradicate tumor cells

The implantation of small pieces of human primary lung tumor biopsy tissue into SCID mice results in a viable s.c. xenograft in which the tissue architecture, including tumor-associated leukocytes, tumor cells, and stromal cells, is preserved in a functional state. By monitoring changes in tumor volume, gene expression patterns, cell depletion analysis, and the use of function-blocking Abs, we previously established in this xenograft model that exogenous IL-12 mobilizes human tumor-associated leukocytes to kill tumor cells in situ by indirect mechanisms that are dependent upon IFN-gamma. In this study immunohistochemistry and FACS characterize the early cellular events in the tumor microenvironment induced by IL-12. By 5 days post-IL-12 treatment, the constitutively present human CD45(+) leukocytes have expanded and infiltrated into tumor-rich areas of the xenograft. Two weeks post-treatment, there is expansion of the human leukocytes and complete effacement of the tumor compared with tumor progression and gradual loss of most human leukocytes in control-treated xenografts. Immunohistochemical analyses reveal that the responding human leukocytes are primarily activated or memory T cells, with smaller populations of B cells, macrophages, plasma cells, and plasmacytoid dendritic cells capable of producing IFN-alpha. The predominant cell population was also characterized by FACS and was shown to have a phenotype consistent with a CD4(+) effector memory T cell. We conclude that quiescent CD4(+) effector memory T cells are present within the tumor microenvironment of human lung tumors and can be reactivated by the local and sustained release of IL-12 to proliferate and secrete IFN-gamma, leading to tumor cell eradication.     

Stabilization of HIF-2α Induces sVEGFR-1 Production from Tumor-Associated Macrophages and Decreases Tumor Growth in a Murine Melanoma Model

Macrophage secretion of vascular endothelial growth factor (VEGF) in response to hypoxia contributes to tumor growth and angiogenesis. In addition to VEGF, hypoxic macrophages stimulated with GM-CSF secrete high levels of a soluble form of the VEGF receptor (sVEGFR-1), which neutralizes VEGF and inhibits its biological activity. Using mice with a monocyte/macrophage-selective deletion of hypoxia-inducible factor (HIF)-1α or HIF-2α, we recently demonstrated that the antitumor response to GM-CSF was dependent on HIF-2α-driven sVEGFR-1 production by tumor-associated macrophages, whereas HIF-1α specifically regulated VEGF production. We therefore hypothesized that chemical stabilization of HIF-2α using an inhibitor of prolyl hydroxylase domain 3 (an upstream inhibitor of HIF-2α activation) would increase sVEGFR-1 production from GM-CSF-stimulated macrophages. Treatment of macrophages with the prolyl hydroxylase domain 3 inhibitor AKB-6899 stabilized HIF-2α and increased sVEGFR-1 production from GM-CSF-treated macrophages, with no effect on HIF-1α accumulation or VEGF production. Treatment of B16F10 melanoma-bearing mice with GM-CSF and AKB-6899 significantly reduced tumor growth compared with either drug alone. Increased levels of sVEGFR-1 mRNA, but not VEGF mRNA, were detected within the tumors of GM-CSF- and AKB-6899-treated mice, correlating with decreased tumor vascularity. Finally, the antitumor and antiangiogenic effects of AKB-6899 were abrogated when mice were simultaneously treated with a sVEGFR-1 neutralizing Ab. These results demonstrate that AKB-6899 decreases tumor growth and angiogenesis in response to GM-CSF by increasing sVEGFR-1 production from tumor-associated macrophages. Specific activation of HIF-2α can therefore decrease tumor growth and angiogenesis.     

Knockdown of hypoxia-inducible factor-1alpha in breast carcinoma MCF-7 cells results in reduced tumor growth and increased sensitivity to methotrexate

The hypoxia-inducible factor (HIF) 1alpha is a key regulator of the cellular response to oxygen deprivation. Specific disruption of the HIF-1 pathway is important for exploring its role in tumor biology and developing more efficient weapons to treat cancer. In this study, we stably transfected human breast tumor MCF-7 cells with short hairpin RNA expression vectors targeting HIF-1alpha. After knockdown of HIF-1alpha, hypoxia-induced expression of its target genes such as vascular endothelial growth factor, Glut-1, phosphoglycerate kinase, and P-glycoprotein were markedly attenuated. Moreover, HIF-1alpha knockdown was found to suppress the shift from S-phase to G(1) induced by hypoxia and increase drug sensitivity to methotrexate. The growth rates of HIF1alpha-knockdown tumors were drastically retarded in both subcutaneous and orthotopic xenograft models, which were accompanied by decreased angiogenesis and reduced expression of glucose transporter in tissue sections. These data demonstrate that HIF-1alpha knockdown reduces tumorigenicity of MCF-7 cells and suggest a promising combination of both anti-HIF-1 strategy and traditional chemotherapy to improve cancer treatment.     

Erythropoietin attenuates cachectic events and decreases production of interleukin-6, a cachexia-inducing cytokine

In cancer cachexia, erythropoietin often yields beneficial therapeutic effects by improving patient's metabolic and exercise capacity via an increased erythrocyte count. However, erythropoietin also has counter-regulatory effects against pro-inflammatory cytokines, which are postulated to be mediators of cancer cachexia. We investigated the mechanisms by which erythropoietin improves the cachectic condition. In this study, 100 Units/day of erythropoietin were administered intraperitoneally to BALB/c male mice, carrying a subclone of colon 26 adenocarcinoma, beginning on the day after tumor inoculation and continuing until they died. Erythropoietin administration attenuated the decline in body weight, as well as the decline in fat and muscle weights, of tumor-bearing mice, but improved the survival of cachectic mice. Mice receiving erythropoietin had increased erythrocyte and platelet counts, but significantly decreased white blood cell count. In addition, erythropoietin administration significantly decreased interleukin-6 levels, not only in serum but also in the inoculated tumor. These results indicate that the positive therapeutic effects of erythropoietin on cancer cachexia are due, not only to improving metabolic and exercise capacity via an increased erythrocyte count, but also to attenuation of cachectic manifestations by decreased production of the cachexia-inducing cytokine, interleukin-6.