Crizotinib induces autophagy through inhibition of the STAT3 pathway in multiple lung cancer cell lines

Autophagy is an evolutionarily conserved survival pathway in eukaryote and is frequently upregulated in cancer cells after chemotherapy or targeted therapy. Thus induction of autophagy has emerged as a drug resistance mechanism. In this study, we found that crizotinib induced a high level of autophagy in lung cancer cells through inhibition of STAT3. Ectopic expression of wild-type or constitutive activated STAT3 significantly suppressed the effect of crizotinib on autophagy. Interestingly, crizotinib-mediated inhibition of STAT3 is in a step-wise manner. Firstly it inhibited cytoplasmic STAT3, which leads to the phosphorylation of EIF2A, then inhibited nuclear STAT3, which leads to the downregulation of BCL-2. Cell death induced by crizotinib was greatly enhanced after the inhibition of autophagy by the pharmacological inhibitors or shRNAs against Beclin-1. Moreover, the autophagy inhibitor HCQ significantly augmented the anti-tumor effect of crizotinib in a mouse xenograft model. In conclusion, crizotinib can induce cytoprotective autophagy by suppression of STAT3 in lung cancer cells. Thus, autophagy inhibition represents a promising approach to improve the efficacy of crizotinib in the treatment of targeted lung cancer patients.     

KLF3 is a crucial regulator of metastasis by controlling STAT3 expression in lung cancer

Lung cancer is one of the most common causes of cancer‐related mortality worldwide, which is partially due to its metastasis. However, the mechanism underlying its metastasis remains elusive. In this study, we showed that a low Krüppel‐like factor 3 (KLF3) expression level is correlated with a poor prognosis and TNM stages in clinical patients with lung cancer and further demonstrated that KLF3 expression is downregulated in lung cancer tissues compared with adjacent normal samples. In addition, bioinformatics analysis results showed that KLF3 expression is related to lung cancer epithelial‐mesenchymal transition (EMT). In vitro and in vivo experiments also showed that KLF3 silencing promotes lung cancer EMT and enhances lung cancer metastasis. More importantly, bioinformatics analysis and in vitro experiments indicated that the role of KLF3 in lung cancer metastasis is dependent on the STAT3 signaling pathway. Overall, our data indicated the crucial function of KLF3 in lung cancer metastasis and suggested opportunities to improve the therapy of patients with lung cancer.     

Mesenchymal stem cells enhance lung cancer initiation through activation of IL-6/JAK2/STAT3 pathway

Background

The role of mesenchymal stem cells (MSCs) and IL-6 in lung cancer has not been well-addressed. We aimed to determine if MSCs can enhance the ability of tumor initiation of lung cancer cells, and link MSCs with activation of the IL-6/JAK2/STAT3 signaling pathway.

Materials and methods

Lung cancer cell lines A549 and CL1-5 were directly or indirectly cocultured with MSCs. Spheres were defined as cell colonies with >50% area showing 3-dimensional structure and blurred cell margins. Cells without and with MSCs were injected into NOD/SCID mice. The percentage of tumor formation was determined. The influence of the IL-6/JAK2/STAT3 signaling pathway in cancer cell sphere formation and tumor growth were investigated.

Results

A very small number of lung cancer cells, when mixed with otherwise non-tumorigenic MSCs, obtained de novo tumorigenicity when injected subcutaneously and allowed to form a tumor xenograft. Secretion of IL-6 from MSCs increased activation of the JAK2/STAT3 pathway in cancer cells, and enhanced sphere formation and tumor initiation. A reduced capacity of tumor formation of A549 and CL1-5 lung cancer cells when IL-6 was inhibited in MSCs or STAT3 was silenced in A549 and CL1-5 admixed with MSCs.

Conclusions

Culture of A549 or CL1-5 lung cancer cells with MSCs increased sphere formation, drug resistance, and overexpression of pluripotency markers through activation of the IL-6/JAK2/STAT3 pathway. MSCs enhanced the capability of A549 and CL1-5 lung cancer cells to form tumors in immunodeficient mice. Blockade of the IL-6/JAK2/STAT3 pathway attenuated the capability of A549 and CL1-5 cells to form tumors.     

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。     

Alternative implication of CXCR4 in JAK2/STAT3 activation in small cell lung cancer

Small cell lung cancer (SCLC) is an aggressive, rapidly metastasising tumour. Previously, we demonstrated the influence of CXCL12–CXCR4 interaction on processes involved in metastasis and chemoresistance in SCLC. We show here that STAT3 is expressed in both primary SCLC tumour tissues and SCLC cell lines. We investigated the function of STAT3 upon CXCL12 stimulation in SCLC cell lines. Small cell lung cancer cell lines present constitutive phosphorylation of STAT3, and in the reference cell lines NCI-H69 and NCI-H82 constitutive phosphorylation was further increased by CXCL12 stimulation. Further investigating this signalling cascade, we showed that it involves interactions between CXCR4 and JAK2 in both cell lines. However CXCL12-induced adhesion to VCAM-1 could be completely inhibited by the JAK2 inhibitor AG490 only in NCI-H82. Furthermore, CXCR4 antagonist but not AG490 inhibited cell adhesion whereas both antagonisms were shown to inhibit growth of the cells in soft agar, indicating the central involvement of this signalling in anchorage-independent growth of SCLC cells. Most interestingly, while using primary tumour material, we observed that in contrast to non-small-cell lung cancer samples from primary tumour tissues, all analysed samples from SCLC were strongly positive for tyrosine-phosphorylated STAT3. Taken together, these data indicate that STAT3 is constitutively phosphorylated in SCLC and is important in SCLC growth and spreading thus presenting an interesting target for therapy.     

Flubendazole elicits anti‐metastatic effects in triple‐negative breast cancer via STAT3 inhibition

Tumor metastasis remains the cause of 90% of cancer‐related deaths. Cancer stem cells (CSC) are thought to be responsible for the aggressive and metastatic nature of triple‐negative breast cancers (TNBC), and new therapeutic strategies are being devised to target them. Flubendazole (FLU) is a widely used anthelmintic agent that also exhibits anticancer activity in several cancer types. The aim of this study was to characterize the mechanism of action of FLU on breast cancer stem cell (BCSC)‐like properties and metastasis in TNBC. FLU treatment caused a significant induction of apoptosis, accompanied by G2/M phase accumulation, caspase‐3/‐7 activation and the dysregulation of STAT3 activation in TNBC cells. The latter phenomenon was associated with impairment of cancer stem‐like traits, concomitant with a reduction in the CD24^low/CD44^high, CD24^high/CD49f^high subpopulation, ALDH1 activity and mammosphere formation. The BCSC‐enriched populations exhibited enhanced metastasis with higher STAT3 activation, while FLU administration inhibited tumor growth, angiogenesis and lung and liver metastasis, coinciding with decreased MMP‐2 and MMP‐9 levels in circulating blood. FLU kills not only rapid proliferating tumor cells but also effectively eradicates BCSC‐like cells in vitro and in vivo. Our findings warrant further investigation of FLU as a treatment for metastatic TNBC.     

Hispidulin suppresses cell growth and metastasis by targeting PIM1 through JAK2/STAT3 signaling in colorectal cancer

Colorectal cancer (CRC) accounts for over 600 000 deaths annually worldwide. The current study aims to evaluate the value of proto‐oncogene PIM1 as a therapeutic target in CRC and investigate the anticancer activity of hispidulin, a naturally occurring phenolic flavonoid compound, against CRC. Immunohistochemistry analysis showed that PIM1 was upregulated in CRC tissue. The role of PIM1 as an oncogene was evidenced by the fact that PIM1 knockdown inhibits cell growth, induces apoptosis, and suppresses invasion. Our results showed that hispidulin exerts antitumor activity in CRC through inhibiting the expression of PIM1. Moreover, our findings revealed that hispidulin downregulated the expression of PIM1 by inhibiting JAK2/STAT3 signaling by generating reactive oxygen species. Furthermore, our in vivo studies showed that hispidulin can significantly inhibit tumor growth and metastasis in CRC. Collectively, our results provide an experimental basis for trialing hispidulin in CRC treatment. PIM1 can be considered a potential therapeutic target in CRC.     

Prevention of cigarette smoke–induced lung tumors in mice by budesonide,phenethyl isothiocyanate,and N‐acetylcysteine

Lung cancer is the most important cause of death among neoplastic diseases worldwide, and cigarette smoke (CS) is the major risk factor for cancer. Complementarily to avoidance of exposure to CS, chemoprevention will lower the risk of cancer in passive smokers, ex‐smokers, and addicted current smokers who fail to quit smoking. Unfortunately, chemoprevention clinical trials have produced disappointing results to date and, until recently, a suitable animal model evaluating CS carcinogenicity was not available. We previously demonstrated that mainstream CS induces a potent carcinogenic response when exposure of mice starts at birth. In the present study, neonatal mice (strain H) were exposed to CS for 120 consecutive days, starting at birth. The chemopreventive agents budesonide (2.4 mg/kg diet), phenethyl isothiocyanate (PEITC, 1,000 mg/kg diet), and N‐acetyl‐L‐cysteine (NAC, 1,000 mg/kg body weight) were administered orally according to various protocols. The experiment was stopped after 210 days. Exposure to CS resulted in a high incidence and multiplicity of benign lung tumors and in significant increases of malignant lung tumors and other histopathological alterations. All three chemopreventive agents, administered to current smokers after weaning, were quite effective in protecting both male and female mice from CS pulmonary carcinogenicity. When given to ex‐smokers after withdrawal of exposure to CS, the protective capacity of budesonide was unchanged, while PEITC lost part of its cancer chemopreventive activity. In conclusion, the proposed experimental model provides convincing evidence that it is possible to prevent CS‐induced lung cancer by means of dietary and pharmacological agents.     

Gastrin acting on the cholecystokinin2 receptor induces cyclooxygenase-2 expression through JAK2/STAT3/PI3K/Akt pathway in human gastric cancer cells

Gastrin, cholecystokinin2 receptor (CCK2R), and cyclooxygenase-2 (COX-2) have been implicated in the carcinogenesis and progression of gastric cancer. Our study demonstrated that antagonist or siRNA against CCK2R blocked amidated gastrin (G17)-induced activation of STAT3 and Akt in gastric cancer cell lines. G17-increased COX-2 expression and cell proliferation were effectively blocked by CCK2R antagonist and inhibitors of JAK2 and PI3K. In addition, knockdown of STAT3 expression significantly attenuated G17-induced PI3K/Akt activation, COX-2 expression, and cell proliferation. These results suggest that CCK2R-mediated COX-2 up-regulation via JAK2/STAT3/PI3K/Akt pathway is involved in the proliferative effect of G17 on human gastric cancer cells.     

隐丹参酮联合顺铂抗非小细胞肺癌NCI-H1975细胞JAK2/STAT3机制研究

目的 探讨隐丹参酮与顺铂联合作用于非小细胞肺癌NCI-H1975细胞具有协同抗肿瘤效应及其可能的分子机制。方法 实验分为对照组、隐丹参酮组、顺铂组及隐丹参酮和顺铂联合用药组(以下简称联合用药组),CCK-8法测定细胞增殖抑制率,流式细胞术测定细胞存活率及凋亡,免疫印迹技术检测用药后凋亡蛋白、抗凋亡分子蛋白表达水平、JAK2/STAT3蛋白及其磷酸化表达水平,激光共聚焦/荧光素酶检测STAT3细胞定位和转录活性。结果 (1)在各时间点,单药组及联合用药组存活率均低于对照组(P＜0.05);与对照组及单药组相比,联合用药组在各时间点均抑制NCI-H1975细胞增殖活性(P＜0.05);(2)联合用药组作用于NCI-H1975细胞24 h后,抗凋亡蛋白Bcl-2、Survivin表达水平下降(P＜0.01),Caspase3和Caspase9活性提高(P＜0.01),p-STAT3及p-JAK2表达水平下降(P＜0.01);(3)联合用药组作用于NCI-H1975细胞后,激光共聚焦检测提示细胞核内STAT3明显减少,核内STAT3活性降低。结论 隐丹参酮联合顺铂作用于非小细胞肺癌NCI-H1975细胞能发挥协同抗肿瘤作用,其机制与抑制信号通路JAK2/STAT3磷酸化表达水平有关。