AZD1480 delays tumor growth in a melanoma model while enhancing the suppressive activity of myeloid-derived suppressor cells

AZD1480 is a potent, competitive small-molecule inhibitor of JAK1/2 kinase which inhibits STAT3 phosphorylation and tumor growth. Here we investigated the effects of AZD1480 on the function of different immune cell populations in a melanoma model. When MO4 tumor-bearing mice were treated with AZD1480 we observed a strong inhibition of tumor growth as well as a prolonged survival. Moreover, a significant decrease in the percentage of myeloid-derived suppressor cells (MDSCs) was observed after treatment with AZD1480. However, AZD1480 enhanced the suppressive capacity of murine MDSCs while at the same time impairing the proliferative as well as the IFN-γ secretion capacity of murine T cells. The addition of AZD1480 to co-cultures of human MDSCs and T cells does not affect the suppressive activity of MDSCs but it does reduce the IFN-γ secretion and the proliferative capacity of T cells. We showed that although AZD1480 has the ability to delay the tumor growth of MO4 tumor-bearing mice, this drug has detrimental effects on several aspects of the immune system. These data indicate that systemic targeting of the JAK/STAT pathway by JAK1/2 inhibition can have divergent effects on tumor growth and anti-tumor immune responses.     

Inhibition of STAT3 with orally active JAK inhibitor,AZD1480, decreases tumor growth in Neuroblastoma and Pediatric Sarcomas In vitro and In vivo

The IL-6/JAK/STAT pathway is a key signal transduction pathway implicated in the pathogenesis of many human cancers, suggesting that kinase inhibitors targeting JAK/STAT3 may have a broad spectrum of antitumor activity. AZD1480, a pharmacological JAK1/2 inhibitor, exhibits anti-tumor potency in multiple adult malignancies. To evaluate the efficacy of inhibition of JAK/STAT3 signal transduction pathway we assessed the activity of AZD1480 in pediatric malignancies using preclinical models of three highly malignant pediatric solid tumors: neuroblastoma (NB), rhabdomyosarcoma (RMS) and the Ewing Sarcoma Family Tumors (ESFT). In this study, we employed panels of biomedical and biological experiments to evaluate the in vitro and in vivo activity of AZD1480 in NB, RMS and ESFT. Our data indicate that AZD1480 blocks endogenous as well as IL-6 induced STAT3 activation. AZD1480 decreases cell viability in 7/7NB, 7/7RMS and 2/2 ESFT cell lines (median EC₅₀ is 1.5 μM, ranging from 0.36-5.37μM). AZD1480 induces cell growth inhibition and caspase-dependent apoptosis in vitro and decreases expression of STAT3 target genes, including cell cycle regulators CyclinD1, 3 and CDC25A, anti-apoptotic genes Bcl-2 and survivin, the metastasis-related factor TIMP-1 and c-Myc. In vivo studies showed AZD1480 significantly decreased tumor growth and prolonged overall survival in tumor-bearing mice. Tumors from AZD1480-treated mice showed inhibition of activated STAT3 as well as decreased expression of STAT3 downstream targets. Our study provides strong evidence of the anti-tumor growth potency of JAK inhibitor AZD1480 in pediatric solid tumors, providing proof-of principle that inhibition of the JAK/STAT3 signal transduction could be a promising therapeutic target for high-risk pediatric solid tumors.     

Modulating Antiangiogenic Resistance by Inhibiting the Signal Transducer and Activator of Transcription 3 Pathway in Glioblastoma

Determining the mechanism of treatment failure of VEGF signaling inhibitors for malignant glioma patients would provide insight into approaches to overcome therapeutic resistance. In this study, we demonstrate that human glioblastoma tumors failing bevacizumab have an increase in the mean percentage of p-STAT3-expressing cells compared to samples taken from patients failing non-antiangiogenic therapy containing regimens. Likewise, in murine xenograft models of glioblastoma, the mean percentage of p-STAT3-expressing cells in the gliomas resistant to antiangiogenic therapy was markedly elevated relative to controls. Administration of the JAK/STAT3 inhibitor AZD1480 alone and in combination with cediranib reduced the infiltration of VEGF inhibitor-induced p-STAT3 macrophages. Thus, the combination of AZD1480 with cediranib markedly reduced tumor volume, and microvascular density, indicating that up regulation of the STAT3 pathway can mediate resistance to antiangiogenic therapy and combinational approaches may delay or overcome resistance.     

通讯作者：花宝金(1964-),男,黑龙江人,主任医师,博士研究生导师,研究方向: 中医药防治肿瘤复发转移。稿件联系人：蒋树龙,E-mail:jnsljiang@163.com。联系电话：13506383007。

【】　目的：总结JAK2/STAT3/SOCS3信号通路的作用特点及其与肿瘤转移潜能的最新研究进展。方法：应用PubMed及CNKI期刊全文数据库,以“JAK2、STAT3、SOCS3、肿瘤和信号转导”为关键词,检索2000-01~2013-10的相关文献,共检索到英文文献343篇,中文文献66篇。文献纳入标准：１)JAK2/STAT3/SOCS3信号通路的生物学特征及作用机制;２)JAK2/STAT3/SOCS3信号通路与肿瘤微环境的关系及其与肿瘤侵袭转移之间的内在联系。根据纳入标准符合条件的30篇。结果：JAK2/STAT3信号通路的激活参与了肿瘤发生、发展、侵袭和转移等多个环节;SOCS3负性调控JAK2/STAT3通路,从而抑制肿瘤的增殖和生长;STAT3信号通路的激活促成了肿瘤炎性微环境的形成,参与了肿瘤血管生成、上皮间质转化(EMT)、细胞外基质(ECM)降解等多个环节,在肿瘤的侵袭和转移过程中发挥重要作用。结论：JAK2/STAT3/SOCS3信号通路与肿瘤转移密切相关,针对JAK2/STAT3/SOCS3信号通路多靶点干预是肿瘤防治研究的一个新方向。     

Effect of AZD1480 in an epidermal growth factor receptor-driven lung cancer model

Objective

STAT3 plays a vital role in inducing and maintaining a pro-carcinogenic inflammatory microenvironment and is reported to be a critical mediator of the oncogenic effects of EGFR mutations. STAT3 activation is mediated through JAK family kinases. We investigated the effect of the JAK1/2 inhibitor AZD1480 on lung tumors induced by an activating EGFR mutation.

Materials and methods

Three EGFR tyrosine kinase inhibitor-resistant cell lines (RPC-9, PC-9/Van-R and PC-9/ER3) established from PC-9 harboring an EGFR exon19 deletion mutation were used. Growth inhibition was measured using an MTT assay. Effects of AZD1480 were also evaluated in the xenograft model and in the EGFR transgenic mice model. Protein expressions were assessed by immunoblotting and immunohistochemistry. Group differences were compared using Student's t-test. To evaluate the efficacy of AZD1480 on survival, AZD1480 or vehicle was administered orally from 7 weeks of age of the transgenic mice. Overall survival curves were calculated using the Kaplan–Meier method.

Results

The sensitivities of resistant and parent cells to AZD1480 were similar in vitro. AZD1480 (30 or 50 mg/kg/day, per os) reduced angiogenesis and revealed significant tumor regression in a mouse xenograft model. Subsequently, the transgenic mice were treated with AZD1480 (30 mg/kg/day) or vehicle alone. The numbers of lung tumors (long axis exceeding 1 mm) in the AZD1480-treated group and control group were 0.37 ± 0.18 and 2.25 ± 0.53 (p < 0.001), respectively. AZD1480 treatment suppressed pSTAT3, pJAK1, pJAK2 and angiogenesis. The median survival time in the AZD1480-treated group (217 days) was significantly greater than that in the control group (106 days) (log-rank test, p < 0.0001).

Conclusion

AZD1480 may be effective against lung tumors driven by an activating EGFR mutation.     

JAK–STAT inhibition impairs K-RAS-driven lung adenocarcinoma progression

Oncogenic K-RAS has been difficult to target and currently there is no K-RAS-based targeted therapy available for patients suffering from K-RAS-driven lung adenocarcinoma (AC). Alternatively, targeting K-RAS-downstream effectors, K-RAS-cooperating signaling pathways or cancer hallmarks, such as tumor-promoting inflammation, has been shown to be a promising therapeutic strategy. Since the JAK–STAT pathway is considered to be a central player in inflammation-mediated tumorigenesis, we investigated here the implication of JAK–STAT signaling and the therapeutic potential of JAK1/2 inhibition in K-RAS-driven lung AC. Our data showed that JAK1 and JAK2 are activated in human lung AC and that increased activation of JAK–STAT signaling correlated with disease progression and K-RAS activity in human lung AC. Accordingly, administration of the JAK1/2 selective tyrosine kinase inhibitor ruxolitinib reduced proliferation of tumor cells and effectively reduced tumor progression in immunodeficient and immunocompetent mouse models of K-RAS-driven lung AC. Notably, JAK1/2 inhibition led to the establishment of an antitumorigenic tumor microenvironment, characterized by decreased levels of tumor-promoting chemokines and cytokines and reduced numbers of infiltrating myeloid derived suppressor cells, thereby impairing tumor growth. Taken together, we identified JAK1/2 inhibition as promising therapy for K-RAS-driven lung AC.     

JAK2信号通路与肿瘤血管新生

JAK2是 JAK 家族的成员之一,JAK2与 STAT 家族的多个成员共同构成多条信号转导通路,如JAK2／STAT3、JAK2／STAT5等。JAK2／STATs 信号通路通过配体和细胞表面的受体结合而诱导受体二聚化,并相互磷酸化,从而激活 JAK。激活了的 JAK2／STAT 信号通路参与了肿瘤的发生、发展、血管新生、侵袭和转移等多个环节。研究表明肿瘤细胞中活化的 JAK2／STATs 信号通路主要是通过上调多种血管生成相关因子如血管内皮生长因子（VEGF）、环氧化酶－2（COX －2）等表达来促进肿瘤血管生成,IFN －α、SHP －1、SOCS通过 JAK2／STATs 通路下调肿瘤细胞促血管生成因子表达,抑制肿瘤血管生成。本文就 JAK2信号通路与肿瘤血管新生作一综述。     

Erythropoietin promotes the growth of pituitary adenomas by enhancing angiogenesis

rhEPO is frequently used in clinical practice to treat anemia. However, recently rhEPO has been reported to accelerate tumor growth, progression and metastasis. Many pituitary adenoma patients, particularly those with macroprolactinomas, tend to have anemia and may need rhEPO therapy. To date, whether rhEPO has deleterious effects on pituitary adenomas has not been defined. Here we demonstrated for the first time that human pituitary adenomas are EPOR negative tumors and rhEPO accelerated the tumor growth of MMQ pituitary adenoma xenografts via enhancement of angiogenesis in vivo, whereas rhEPO displayed no direct effect on MMQ cells in vitro. Our mechanistic study showed that rhEPO administration increased phosphorylation of JAK2, STAT3 and VEGF expression in human umbilical vein endothelial cells (HUVECs) in vitro and in MMQ cell xenografts in vivo. Furthermore, VEGF inhibitor attenuated rhEPO induced angiogenesis and delayed tumor growth in MMQ pituitary adenoma xenografts in vivo. JAK2 inhibitor AG490 attenuated EPO induced HUVECs proliferation, phosphorylation of JAK2, STAT3 and VEGF upregulation in vitro and inhibited EPO induced vessel formation in Chicken chorioallantoic membrane (CAM) angiogenesis model in vivo. These results suggest that rhEPO administration may promote the growth of pituitary adenomas by enhancing angiogenesis through EPO-JAK2-STAT3-VEGF signaling pathway. rhEPO should be used with caution in anemia patients bearing pituitary adenoma due to its potential deleterious effects.     

6-Bromoindirubin-3'-oxime inhibits JAK/STAT3 signaling and induces apoptosis of human melanoma cells

STAT3 is persistently activated and contributes to malignant progression in various cancers. Janus activated kinases (JAK) phosphorylate STAT3 in response to stimulation by cytokines or growth factors. The STAT3 signaling pathway has been validated as a promising target for development of anticancer therapeutics. Small-molecule inhibitors of JAK/STAT3 signaling represent potential molecular-targeted cancer therapeutic agents. In this study, we investigated the role of JAK/STAT3 signaling in 6-bromoindirubin-3'-oxime (6BIO)-mediated growth inhibition of human melanoma cells and assessed 6BIO as a potential anticancer drug candidate. We found that 6BIO is a pan-JAK inhibitor that induces apoptosis of human melanoma cells. 6BIO directly inhibited JAK-family kinase activity, both in vitro and in cancer cells. Apoptosis of human melanoma cells induced by 6BIO was associated with reduced phosphorylation of JAKs and STAT3 in both dose- and time-dependent manners. Consistent with inhibition of STAT3 signaling, expression of the antiapoptotic protein Mcl-1 was downregulated. In contrast to the decreased levels of phosphorylation of JAKs and STAT3, phosphorylation levels of the Akt and mitogen-activated protein kinase (MAPK) signaling proteins were not inhibited in cells treated with 6BIO. Importantly, 6BIO suppressed tumor growth in vivo with low toxicity in a mouse xenograft model of melanoma. Taken together, these results show that 6BIO is a novel pan-JAK inhibitor that can selectively inhibit STAT3 signaling and induces tumor cell apoptosis. Our findings support further development of 6BIO as a potential anticancer therapeutic agent that targets JAK/STAT3 signaling in tumor cells.     

Myeloid-derived suppressor cells: A novel therapeutic target

Immunotherapy and angiogenic inhibitors, used alone or in combination with chemotherapy, represent two promising cancer treatment programs. Each is limited by myeloid-derived suppressor cells (MDSCs), which accumulate in tumor-bearing hosts. MDSCs inhibit effector T-cell function and thus prevent the formation and execution of an effective antitumor immune response. Recently reported studies have shown that MDSCs also function to promote tumordependent angiogenesis as well as tumor metastasis, and to provide tumor resistance to antiangiogenic drugs. Insights into tumor-imposed dynamic changes in bone marrow function and myeloid cell development should fuel novel drug developments and novel applications of drugs currently in use. Such insights suggest that multitargeted receptor tyrosine kinase inhibitors, such as sunitinib, may be useful adjunctive agents for use in immunotherapy trials treating several tumor types.     

SU5416 and SU6668 attenuate the angiogenic effects of radiation-induced tumor cell growth factor production and amplify the direct anti-endothelial action of radiation in vitro

In recent decades, radiation research has concentrated primarily on the cancer cell compartment. Much less is known about the effect of ionizing radiation on the endothelial cell compartment and the complex interaction between tumor cells and their microenvironment. Here we report that ionizing radiation is a potent antiangiogenic agent that inhibits endothelial cell survival, proliferation, tube formation and invasion. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor were able to reduce the radiosensitivity of endothelial cells. Yet, it is also found that radiation induces angiogenic factor production by tumor cells that can be abrogated by the addition of antiangiogenic agents. Receptor tyrosine kinase inhibitors of Flk-1/KDR/VEGFR2, FGFR1 and PDGFR beta, SU5416, and SU6668 enhanced the antiangiogenic effects of direct radiation of the endothelial cells. In a coculture system of PC3 prostate cancer cells and endothelial cells, isolated irradiation of the PC3 cells enhanced endothelial cell invasiveness through a Matrigel matrix, which was inhibited by SU5416 and SU6668. Furthermore, ionizing radiation up-regulated VEGF and basic fibroblast growth factor in PC3 cells and VEGFR2 in endothelial cells. Together these findings suggest a radiation-inducible protective role for tumor cells in the support of their associated vasculature that may be down-regulated by coadministration of angiogenesis inhibitors. These results rationalize concurrent administration of angiogenesis inhibitors and radiotherapy in cancer treatment.