Background:Although clinical studies have shown promise for targeting PD1/PDL1 signaling in non–small cell lung cancer (NSCLC), the regulation of PDL1 expression is poorly understood. Here, we show that PDL1 is regulated by p53 via miR-34.Methods:p53 wild-type and p53-deficient cell lines (p53–/– and p53+/+ HCT116, p53-inducible H1299, and p53-knockdown H460) were used to determine if p53 regulates PDL1 via miR-34. PDL1 and miR-34a expression were analyzed in samples from patients with NSCLC and mutated p53 vs wild-type p53 tumors from The Cancer Genome Atlas for Lung Adenocarcinoma (TCGA LUAD). We confirmed that PDL1 is a direct target of miR-34 with western blotting and luciferase assays and used a p53R172HΔg/+K-rasLA1/+ syngeneic mouse model (n = 12) to deliver miR-34a–loaded liposomes (MRX34) plus radiotherapy (XRT) and assessed PDL1 expression and tumor-infiltrating lymphocytes (TILs). A two-sided t test was applied to compare the mean between different treatments.Results:We found that p53 regulates PDL1 via miR-34, which directly binds to the PDL1 3’ untranslated region in models of NSCLC (fold-change luciferase activity to control group, mean for miR-34a = 0.50, SD = 0.2, P < .001; mean for miR-34b = 0.52, SD = 0.2, P = .006; and mean for miR-34c = 0.59, SD = 0.14, and P = .006). Therapeutic delivery of MRX34, currently the subject of a phase I clinical trial, promoted TILs (mean of CD8 expression percentage of control group = 22.5%, SD = 1.9%; mean of CD8 expression percentage of MRX34 = 30.1%, SD = 3.7%, P = .016, n = 4) and reduced CD8+PD1+ cells in vivo (mean of CD8/PD1 expression percentage of control group = 40.2%, SD = 6.2%; mean of CD8/PD1 expression percentage of MRX34 = 20.3%, SD = 5.1%, P = .001, n = 4). Further, MRX34 plus XRT increased CD8+ cell numbers more than either therapy alone (mean of CD8 expression percentage of MRX34 plus XRT to control group = 44.2%, SD = 8.7%, P = .004, n = 4). Finally, miR-34a delivery reduced the numbers of radiation-induced macrophages (mean of F4-80 expression percentage of control group = 52.4%, SD = 1.7%; mean of F4-80 expression percentage of MRX34 = 40.1%, SD = 3.5%, P = .008, n = 4) and T-regulatory cells.Conclusions:We identified a novel mechanism by which tumor immune evasion is regulated by p53/miR-34/PDL1 axis. Our results suggest that delivery of miRNAs with standard therapies, such as XRT, may represent a novel therapeutic approach for lung cancer.

The i.v. delivery of an oncolytic herpes virus may achieve effective infection, oncolysis, and transgene expression at distant tumor sites. This approach to systemic therapy combining oncolysis with IL-12 immune stimulation led to significantly improved survival in animals with disseminated SCC.

The cell membrane receptor ErbB-2 migrates to the nucleus. However, the mechanism of its nuclear translocation is unclear. Here, we report a novel mechanism of its nuclear localization that involves interaction with the transport receptor importin ␤1, nuclear pore protein Nup358, and a host of players in endocytic internalization. Knocking down importin ␤1 using small interfering RNA oligonucleotides or inactivation of small GTPase Ran by RanQ69L, a dominant-negative mutant of Ran, causes a nuclear transport defect of ErbB-2. Mutation of a putative nuclear localization signal in ErbB-2 destroys its interaction with importin ␤1 and arrests nuclear translocation, while inactivation of nuclear export receptor piles up ErbB-2 within the nucleus. Additionally, blocking of internalization by a dominant-negative mutant of dynamin halts its nuclear localization. Thus, the cell membrane-embedded ErbB-2, through endocytosis using the endocytic vesicle as a vehicle, importin ␤1 as a driver and Nup358 as a traffic light, migrates from the cell surface to the nucleus. This novel mechanism explains how a receptor tyrosine kinase on the cell surface can be translocated into the nucleus. This pathway may serve as a general mechanism to allow direct communication between cell surface receptors and the nucleus, and our findings thus open a new era in understanding direct trafficking between the cell membrane and nucleus.Despite a number of recent reports on translocation of receptor tyrosine kinases (RTKs) to the nucleus (reviewed in references 8 and 52), the mechanism of how RTKs travel from the cell surface to the nucleus is still unknown. Transmembrane receptors serve as sensors, recognizing the growth factor in the extracellular environment and conveying the message through the signaling cascade to the nucleus. However, some RTKs, like the epidermal growth factor receptor (EGFR) family members (27,29,32,36,37,49,54), fibroblast growth factor receptor 1 (FGFR1) and FGFR3 and their splice variants (23,31,40,43,44,45,57), insulin receptor (41), and vascular endothelium growth factor receptor Flk1/KDR (13,21,34), are known to migrate to the nucleus either intact or as a fragment of proteolytic cleavage, interestingly, with or without the corresponding ligand. These nuclear RTKs have been shown to act as transcription factors (27,36,49,54) for genes like Cyclin D1 (27), FGF2 (38), and COX-2 (49) and modulators for induction of c-jun and cyclin D1 (40). Additionally, nuclear matrix binding of FGFR1 and insulin receptor (41, 45) has been shown to strategically position the receptors for involvement in the regulation of gene expression. More recently, the nuclear EGFR was shown to interact with well-known DNA binding transcriptional factors, such as STAT3 and E2F1, to regulate the expression of inducible nitric oxide synthase and B-Myb (17, 28). Overall, these reports support direct roles for RTKs in the nucleus, which represent a new class of RTK functions.However, the mechanism of their nuclear import is virtually unknown. We repor...

Prostate cancer is the most common cancer in American men and the second leading cause of cancer deaths in this group. We have found that interleukin (IL)-6 protein concentrations are increased approximately 18-fold in clinically localized prostate cancers when compared to normal prostate tissue. Normal and neoplastic prostatic epithelial cells in culture, with the exception of LNCaP cells, secrete IL-6. Addition of exogenous IL-6 to primary epithelial cells in culture or the LNCaP prostate cancer cell line leads to phosphorylation of Stat-3 and increases in net cell proliferation. The concentration of IL-6 receptor is increased eightfold in the prostate cancer tissues and is increased in the cancer cells by immunohistochemistry. The increased expression of IL-6 receptor is correlated with increased proliferation of prostate cancer cells in vivo as assessed by Ki67 immunohistochemistry. These findings strongly support the hypothesis that IL-6 acts as a significant autocrine growth factor in vivo for primary, androgen-dependent prostate cancers.

The PTEN gene encodes a lipid phosphatase that negatively regulates the phosphatidylinositol 3-kinase pathway and is inactivated in a wide variety of malignant neoplasms. High rates of loss of heterozygosity are observed at the 10q23.3 region containing the human PTEN gene in prostate cancer and other human malignancies, but the demonstrated rate of biallelic inactivation of the PTEN gene by mutation or homozygous deletion is significantly lower than the rate of loss of heterozygosity. The transgenic adenocarcinoma of mouse prostate model is a well characterized animal model of prostate cancer. Analysis of prostate cancer progression in transgenic adenocarcinoma of mouse prostate mice bred to Pten ؉/؊ heterozygous mice, coupled with analysis of the Pten gene and protein in the resulting tumors, reveals that haploinsufficiency of the Pten gene promotes the progression of prostate cancer in this model system. This observation provides a potential explanation for the discordance in rates of loss of heterozygosity at 10q23 and biallelic PTEN inactivation observed in prostate cancer and many human malignancies.T he PTEN tumor suppressor gene (also known as MMAC-1) encodes a phosphatase and is inactivated in a wide variety of human malignant neoplasms, including gliomas, melanomas, and carcinomas of the endometrium, kidney, breast, lung, upper respiratory tract, and prostate (1-4). The tumor suppressor activity of PTEN is thought to be primarily due to its ability to dephosphorylate phosphatidylinositol 3,4,5-phosphate at the 3-position and negatively regulate the activity of the phosphatidylinositol 3-kinase pathway (5, 6). A variety of biological effects have been attributed to loss of PTEN activity that are relevant to its role as a tumor suppressor gene, including enhanced cell proliferation (6), decreased apoptosis (5, 6), and increased tumor angiogenesis (7,8). The PTEN gene is also mutated in Cowden syndrome (9), a hereditary neoplastic syndrome characterized by an increased rate of thyroid cancer and breast cancer in affected females. Thus, the PTEN gene is an important tumor suppressor with a wide range of biological activities relevant to tumor progression.The PTEN tumor suppressor gene maps to human chromosome 10q23.3, and this region shows high rates of loss of heterozygosity (LOH) in a variety of human malignancies. Such LOH is usually due to the loss of relatively large areas of one copy of chromosome 10. It is generally believed that in the presence of such LOH the tumor suppressor gene present on the retained chromosome is inactivated by smaller deletions, resulting in homozygous deletion or by mutation. However, for the PTEN gene, the rate of LOH at 10q23.3 is often much higher than the apparent rate of inactivation of the retained PTEN allele. For example, LOH at 10q23.3 has been detected in 15-49% of clinically localized human prostate cancers, whereas mutation or homozygous deletion of the PTEN gene is detected in less than 10% of these same cases (4, 10-15). Similarly, LOH at 10q23 is present in m...