cucurbitacin-i and Breast-Neoplasms

Immunotherapy for cancer treatment is achieved through the activation of competent immune effector cells and the inhibition of immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs). Although MDSCs have been shown to contribute to breast cancer development, the mechanism underlying MDSC-mediated immunosuppression is unclear. We have identified a poorly differentiated MDSC subset in breast cancer-suppressing T cell function through STAT3-dependent IDO upregulation. In this study we investigated the mechanisms underlying aberrant expression of IDO in MDSCs. MDSCs were induced by coculturing human CD33(+) myeloid progenitors with MDA-MB-231 breast cancer cells. Increased STAT3 activation in MDSCs was correlated with activation of the noncanonical NF-κB pathway, including increased NF-κB-inducing kinase (NIK) protein level, phosphorylation of cytoplasmic inhibitor of NF-κB kinase α and p100, and RelB-p52 nuclear translocation. Blocking STAT3 activation with the small molecule inhibitor JSI-124 significantly inhibited the accumulation of NIK and IDO expression in MDSCs. Knockdown of NIK in MDSCs suppressed IDO expression but not STAT3 activation. RelB-p52 dimers were found to directly bind to the IDO promoter, leading to IDO expression in MDSCs. IL-6 was found to stimulate STAT3-dependent, NF-κB-mediated IDO upregulation in MDSCs. Furthermore, significant positive correlation between the numbers of pSTAT3(+) MDSCs, IDO(+) MDSCs, and NIK(+) MDSCs was observed in human breast cancers. These results demonstrate a STAT3/NF-κB/IDO pathway in breast cancer-derived MDSCs, which provides insight into understanding immunosuppressive mechanisms of MDSCs in breast cancer.

Topics: Active Transport, Cell Nucleus; Breast Neoplasms; Cell Nucleus; Female; Gene Expression Regulation, Neoplastic; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interleukin-6; Myeloid Cells; Neoplasm Proteins; NF-kappaB-Inducing Kinase; Protein Serine-Threonine Kinases; STAT3 Transcription Factor; Transcription Factor RelB; Triterpenes; Up-Regulation

Active cancer-associated fibroblasts (CAFs), which constitute the most preponderant cell type in breast tumors, contribute actively to all aspects of cancer progression, stimulate recurrence, and restrain drug sensitivity. In the present study, we tested the effect of the selective JAK/STAT3 inhibitor cucurbitacin I (JSI-124) on active breast CAFs. We have shown that JSI-124 at non-cytotoxic concentration (20 nM) can inhibit the IL-6/STAT3/NF-κB positive feedback loop in breast myofibroblasts, which enabled persistent inactivation of these cells. Interestingly, JSI-124 treatment suppressed the paracrine promotion of the epithelial-to-mesenchymal transition (EMT) process and the pro-migratory/-invasive and -proliferative effects of CAFs on breast cancer cells in vitro. Similarly, JSI-124 inhibited the capacity of CAF cells in promoting tumor growth, EMT, stemness as well as angiogenesis in orthotopic humanized breast cancer tumors. Together, these findings indicate that JSI-124-dependent inhibition of STAT3 could be of great therapeutic value for the treatment of breast cancer through targeting cancer cells as well as their growth supportive stromal fibroblasts and blood vessels. This could pave the path to developing a precise CAF-targeted anticancer therapy.

Topics: Animals; Breast Neoplasms; Cancer-Associated Fibroblasts; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Feedback, Physiological; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Mice; NF-kappa B; STAT3 Transcription Factor; Triterpenes; Xenograft Model Antitumor Assays

Breast cancer (BC) is the most frequently diagnosed type of cancer all over the world. Angiogenesis, a physiological or pathological process characterized by the sprouting of new blood vessels from existing vessels, plays a vital role in tumor nutrition. In this work, we used JSI-124 (Cucurbitacin I), a selective JAK/STAT3 signaling pathway inhibitor, to investigate the role of STAT3 in tumor angiogenesis of a human BC cell line in vitro. JSI-124 inhibited cell viability, proliferation, adhesion, migration and tube formation of a human BC cell line MDA-MB-468. After transfection with pMXs-Stat3C, a dominant active mutant, the inhibitory effects of JSI-124 on MDA-MB-468 were abolished. Furthermore, JSI-124 reduced the phosphorylation of STAT3. These results suggested that JSI-124 inhibited tumor angiogenesis of the human BC cell line in vitro through the reduction of STAT3 phosphorylation. In addition, JSI-124 could reduce VEGF transcription and secretion, suggesting that JSI-124 is also involved in the inhibition of the VEGF autocrine loop in the tumor microenvironment.

Topics: Autocrine Communication; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Survival; Cell Transformation, Neoplastic; Depression, Chemical; Female; Humans; Molecular Targeted Therapy; Neovascularization, Pathologic; Phosphorylation; Phytotherapy; Signal Transduction; STAT3 Transcription Factor; Transcription, Genetic; Triterpenes; Tumor Microenvironment; Vascular Endothelial Growth Factor A

The small GTPase Rac1 has been widely implicated in mammary tumorigenesis and metastasis. Previous studies established that stimulation of ErbB receptors in breast cancer cells activates Rac1 and enhances motility via the Rac-guanine nucleotide exchange factor P-Rex1. As the Janus tyrosine kinase 2 (Jak2)/signal transducer and activator of transcription 3 (Stat3) pathway has been shown to be functionally associated with ErbB receptors, we asked if this pathway could mediate P-Rex1/Rac1 activation in response to ErbB ligands. Here we found that the anticancer agent cucurbitacin I, a Jak2 inhibitor, reduced the activation of Rac1 and motility in response to the ErbB3 ligand heregulin in breast cancer cells. However, Rac1 activation was not affected by Jak2 or Stat3 RNA interference, suggesting that the effect of cucurbitacin I occurs through a Jak2-independent mechanism. Cucurbitacin I also failed to affect the activation of P-Rex1 by heregulin. Subsequent analysis revealed that cucurbitacin I strongly activates RhoA and the Rho effector Rho kinase (ROCK) in breast cancer cells and induces the formation of stress fibers. Interestingly, disruption of the RhoA-ROCK pathway prevented the inhibitory effect of cucurbitacin I on Rac1 activation by heregulin. Lastly, we found that RhoA activation by cucurbitacin I is mediated by reactive oxygen species (ROS). The ROS scavenger N-acetyl L-cysteine and the mitochondrial antioxidant Mito-TEMPO rescued the inhibitory effect of cucurbitacin I on Rac1 activation. In conclusion, these results indicate that ErbB-driven Rac1 activation in breast cancer cells proceeds independently of the Jak2 pathway. Moreover, they established that the inhibitory effect of cucurbitacin I on Rac1 activity involves the alteration of the balance between Rho and Rac.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Guanine Nucleotide Exchange Factors; Humans; Janus Kinase 2; Ligands; MCF-7 Cells; Neuregulin-1; rac1 GTP-Binding Protein; Reactive Oxygen Species; Receptor, ErbB-3; rho-Associated Kinases; rhoA GTP-Binding Protein; STAT3 Transcription Factor; Stress Fibers; Triterpenes