IL-2 complex recovers steroid-induced inhibition in immunochemotherapy for head and neck cancer

Background: A combination therapy with immune checkpoint inhibitors (ICIs) and platinum-based chemotherapy has become the first-line treatment for recurrent or metastatic head and neck squamous carcinoma (HNSCC). Although steroids are often used as anti-emetic medications during chemotherapy, their adverse effects on immune-combined chemotherapy are unclear in HNSCC.Methods: The effects of dexamethasone on tumor growth and immune cell population were evaluated in a mouse HNSCC model treated with PD-1 blockade combined with cisplatin. The effect of various doses of dexamethasone on cell proliferation, survival, surface markers, IFN-γ production, and antitumor effects in antigen-specific T cells was examined in vitro. The recovery of T cell dysfunction by IL-2 was assessed in vitro and in vivo.Results: In a mouse HNSCC model, dexamethasone showed limited antitumor effects on immunochemotherapy. Dexamethasone decreased the number of T cells and inhibited T cell differentiation into effector and central memory T cells. In the in vitro assessment, dexamethasone induced cell death, limited proliferation, and reduced the reactivity against HNSCC cell lines of antigen-specific T cells in a dose-dependent manner. The expression of inhibitory receptors on T cells was not affected by steroids. This inhibition was recovered by IL-2 and IL-2/anti-IL-2 complexes (IL-2 Cx) in vitro and in vivo, respectively.Conclusion: Our preclinical data indicate that dexamethasone diminishes the antitumor effects of immunochemotherapy in patients with HNSCC. IL-2 Cx recovered the inhibition of antitumor immunity by steroids and might be a potent immune adjuvant for patients who require steroids during PD-1 blockade and chemotherapy.     

Loss-of-Function PTPRD Mutations Lead to Increased STAT3 Activation and Sensitivity to STAT3 Inhibition in Head and Neck Cancer

Background

Protein tyrosine phosphatase receptor type D (PTPRD) is a putative tumor suppressor in several cancers including head and neck squamous cell carcinoma (HNSCC). STAT3 is a frequently hyperactivated oncogene in HNSCC. As STAT3 is a direct substrate of PTPRD, we sought to determine the genetic or epigenetic alterations of PTPRD that contribute to overactive STAT3 in HNSCC.

Methods

We analyzed data from The Cancer Genome Atlas (TCGA) and our previous whole-exome sequencing study and summarized the mutation, methylation, and copy number status of PTPRD in HNSCC and other cancers. In vitro studies involved standard transfection and MTT protocols, as well as methylation-specific PCR.

Results

Our findings indicate that PTPRD mutation, rather than methylation or copy number alteration, is the primary mechanism by which PTPRD function is lost in HNSCC. We demonstrate that overexpression of wild-type PTPRD in HNSCC cells significantly inhibits growth and STAT3 activation while PTPRD mutants do not, suggesting that mutation may lead to loss of function and subsequent hyper-phosphorylation of PTPRD substrates, especially STAT3. Importantly, we determined that HNSCC cells harboring an endogenous PTPRD mutation are more sensitive to STAT3 blockade than PTPRD wild-type cells. We additionally found that PTPRD mRNA expression does not correlate with pSTAT3 expression, suggesting that alterations that manifest through altered mRNA expression, including hypermethylation and gene copy number alterations, do not significantly contribute to STAT3 overactivation in HNSCC.

Conclusion

PTPRD mutation, but not methylation or copy number loss, may serve as a predictive biomarker of sensitivity to STAT3 inhibitors in HNSCC.     

新型抗癌药物分子靶标STAT3的研究进展

信号转导与转录激活因子(STATs)是一类发挥信号转导和转录因子调节作用的蛋白质家族,它们可以作为信号转导分子和转录调节因子参与到细胞因子和生长因子对于正常细胞的调控作用中。STATs的异常激活,特别是STAT3激活,和多种人类恶性肿瘤相关联。相关的分子生物学和药理学模型的研究也已确认STAT3在肿瘤发生中的重要作用,这些工作为抗癌药物研发和治疗癌症提供了新的靶标。此外,结构性活化的STAT3突变体就足以诱导瘤原细胞的转化,并且进一步在体内形成肿瘤。结构性激活的STAT3信号通路常常伴随着一些基因如cyclinD1,c-Myc和Bcl-x的上调,同时也会破坏正常细胞生长与生存的调控机制。体外和体内的实验研究结果也证明,对于STAT3信号通路结构性的阻断可以导致STAT3高表达肿瘤类型中的细胞生长抑制和凋亡。这种已被证实了的肿瘤细胞内的结构性激活和生长存活之间的相互联系,为癌症治疗提供了广阔的应用前景。近年来针对STAT3抑制剂的研究逐渐成为热点,本文就此作一综述。     

The induction of ferroptosis by impairing STAT3/Nrf2/GPx4 signaling enhances the sensitivity of osteosarcoma cells to cisplatin

Recent studies have indicated that promoting ferroptosis is a promising approach to attenuate drug resistance of cancer cells. Hence, this study aimed to induce ferroptosis in osteosarcoma cells, thereby increasing the sensitivity to cisplatin. Osteosarcoma cells MG63 and Saos‐2 were incubated with increasing doses of cisplatin to generate cisplatin‐resistant strains, MG63/DDP and Saos‐2/DDP. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and flow cytometry assays were performed to evaluate cell proliferation and cell death, respectively. Malondialdehyde (MDA), reactive oxygen species (ROS), and lipid oxidation in cells were measured to evaluate the degree of cell ferroptosis. MG63/DDP and Saos‐2/DDP cells showed increased viability and decreased death rate compared with MG63 and Saos‐2 cells, respectively, upon cisplatin treatment. Western blotting analysis indicated that protein levels of p‐STAT3 (Ser727), nuclear factor erythroid 2‐related factor 2 (Nrf2), and glutathione peroxidase 4 (GPx4) in drug‐resistant strains increased significantly in response to cisplatin. Co‐treatment with cisplatin and agonists of ferroptosis, Erastin, and RSL3, remarkably increased MDA, ROS, lipid oxidation, and sensitivity to cisplatin, in MG63/DDP and Saos‐2/DDP cells. Similar results were observed by co‐treatment of cells with cisplatin and a STAT3 inhibitor. The reduction of protein levels of p‐STAT3 (Ser727), Nrf2, and GPx4 in MG63/DDP and Saos‐2/DDP cells resulted in increased ferroptosis and sensitivity to cisplatin. These results indicate that cisplatin‐resistant osteosarcoma cells inhibited ferroptosis after exposure to low doses of cisplatin. However, ferroptosis agonists and STAT3 inhibitor reactivated ferroptosis in the cells and consequently increased sensitivity to cisplatin. This study demonstrates a new approach to attenuate resistance of osteosarcoma to cisplatin in vitro .     

Convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer

BackgroundAberrant activation of STAT3 is frequently encountered and promotes survival, cellular proliferation, migration, invasion and angiogenesis in tumor cell. Convallatoxin, triterpenoid ingredient, exhibits anticancer pharmacological properties.PurposeIn this work, we investigated the anticancer potential of convallatoxin and explored whether convallatoxin mediates its effect through interference with the STAT3 activation in colorectal cancer cells.MethodsIn vitro, the underlying mechanisms of convallatoxin at inhibiting STAT3 activation were investigated by homology modeling and molecular docking, luciferase reporter assay, MTT assay, RT-PCR, Western blotting and immunofluorescence assays. Changes in cellular proliferation, apoptosis, migration, invasion and angiogenesis were analyzed by EdU labeling assay, colony formation assay, flow cytometry assay, wound-healing assay, matrigel transwell invasion assay and tube formation assays. And in vivo, antitumor activity of convallatoxin was assessed in a murine xenograft model of HCT116 cells.ResultsConvallatoxin decreased the viability of colorectal cancer lines. Moreover, convallatoxin reduced the P-STAT3 (T705) via the JAK1, JAK2, and Src pathways and inhibited serine-727 phosphorylation of STAT3 via the PI3K-AKT-mTOR-STAT3 pathways in colorectal cancer cells. Interestingly, we discovered the crosstalk between mTOR and JAK2 in mTOR/STAT3 and JAK/STAT3 pathways, which collaboratively regulated STAT3 activation and convallatoxin play a role in it. Convallatoxin also downregulated the expression of target genes involved cell survival (e.g., Survivin, Bcl-xl, Bcl-2), proliferation (e.g., Cyclin D1), metastasis (e.g., MMP-9), and angiogenesis (e.g., VEGF). Indeed, we found that convallatoxin inhibited tube formation, migration, and invasion of endothelial cells, and inhibited the proliferation. Finally, in vivo observations were confirmed by showing antitumor activity of convallatoxin in a murine xenograft model.ConclusionThe result of the current study show that convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer cells and indicate that convallatoxin could be a valuable candidate for the development of colorectal cancer therapeutic.     

MiR-124 Suppresses Growth of Human Colorectal Cancer by Inhibiting STAT3

Emerging evidence indicate that microRNAs (miRNAs) may play important roles in cancer. Aberrant expression of miRNAs has been frequently identified in different human malignancies, including colorectal cancer (CRC). However, the mechanism by which deregulated miRNAs impact the development of CRC remains largely elusive. In this study, we show that miR-124 is significantly down-regulated in CRC compared to adjacent non-tumor colorectal tissues. MiR-124 suppresses the expression of STAT3 by directly binding to its 3′-untranslated region (3′-UTR). Overexpression of miR-124 led to increased apoptosis of CRC cells and reduced tumor growth in vitro and in vivo. Knocking down STAT3 expression by specific siRNA suppressed the growth of CRC cells in vitro and in vivo, resembling that of miR-124 overexpression. Moreover, overexpression of STAT3 in miR-124-transfected CRC cells effectively rescued the inhibition of cell proliferation caused by miR-124. These data suggest that miR-124 serves as a tumor suppressor by targeting STAT3, and call for the use of miR-124 as a potential therapeutic tool for CRC, where STAT3 is often hyper-activated.     

IL-6, a risk factor for hepatocellular carcinoma: FLLL32 inhibits IL-6-Induced STAT3 phosphorylation in human hepatocellular cancer cells

Hepatocellular carcinoma (HCC) is one of the most common human cancers and the patients' five-year survival rate is very low. Growing evidence indicates that interleukin-6 is a risk factor for HCC. High serum IL-6 may promote HCC development in hepatitis B patients. Therefore, IL-6 could be considered a HCC biomarker and blockade of IL-6 pathway may be a promising therapeutic alternative for HCC. STAT3 is major pathway to mediate signal from IL-6 to the nucleus, where different genes associated with proliferation and apoptosis are regulated. We previous reported that IL-6 induces cell survival upon drug treatment in HCC cells and inhibition of IL-6/STAT3 pathway using anti-IL-6 antibody or STAT3 small-molecule inhibitor LLL12 reduces this effect. Here we summarized the recent studies of IL-6 in HCC and showed another STAT3 small-molecule FLLL32 also blocked IL-6-induced STAT3 activation in HCC cells. FLLL32 is a novel curcumin analogue, which has been described to suppress the constitutive activation of STAT3 in pancreatic and breast cancer cells in vitro and vivo. In this study, we demonstrated that FLLL32 blocked IL-6-induced STAT3 phosphorylation and nuclear translocation.     

Mechanism of STAT3 activation by insulin-like growth factor I receptor

Recent evidence indicates that STAT proteins can be activated by a variety of receptor and non-receptor protein-tyrosine kinases. Unlike cytokine-induced activation of STATs, where JAKs are known to play a pivotal role in phosphorylating STATs, the mechanism for receptor protein-tyrosine kinase-mediated activation of STATs remains elusive. In this study, we investigated the activation of STAT proteins by the insulin-like growth factor I receptor (IGF-IR) in vitro and in vivo and assessed the role of JAKs in the process of activation. We found that STAT3, but not STAT5, was activated in response to IGF-I in 293T cells cotransfected with IGF-IR and STAT expression vectors. Moreover, tyrosine phosphorylation of STAT3, JAK1, and JAK2 was increased upon IGF-I stimulation of endogenous IGF-IR in 293T cells transfected with the respective STAT or JAK expression vector. Supporting the observation in 293T cells, endogenous STAT3 was tyrosine-phosphorylated upon IGF-I stimulation in the muscle cell line C2C12 as well as in various embryonic and adult mouse organs during different stages of development. Dominant-negative JAK1 or JAK2 was able to block the IGF-IR-mediated tyrosine phosphorylation of STAT3 in 293T cells. A newly identified family of proteins called SOCS (suppressor of cytokine signaling), including SOCS1, SOCS2, SOCS3 and CIS, was able to inhibit the IGF-I-induced STAT3 activation as well with varying degrees of potency, in which SOCS1 and SOCS3 appeared to have the higher inhibitory ability. Inhibition of STAT3 activation by SOCS could be overcome by overexpression of native JAK1 and JAK2. We conclude that IGF-I/IGF-IR is able to mediate activation of STAT3 in vitro and in vivo and that JAKs are essential for the process of activation.