PDL1 Regulation by p53 via miR-34.

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 p53(R172HΔ)g/+K-ras(LA1/+) 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.

Therapeutic delivery of miR-200c enhances radiosensitivity in lung cancer.

The microRNA (miR)-200s and their negative regulator ZEB1 have been extensively studied in the context of the epithelial-mesenchymal transition. Loss of miR-200s has been shown to enhance cancer aggressiveness and metastasis, whereas replacement of miR-200 miRNAs has been shown to inhibit cell growth in several types of tumors, including lung cancer. Here, we reveal a novel function of miR-200c, a member of the miR-200 family, in regulating intracellular reactive oxygen species signaling and explore a potential application for its use in combination with therapies known to increase oxidative stress such as radiation. We found that miR-200c overexpression increased cellular radiosensitivity by direct regulation of the oxidative stress response genes PRDX2, GAPB/Nrf2, and SESN1 in ways that inhibits DNA double-strand breaks repair, increase levels of reactive oxygen species, and upregulate p21. We used a lung cancer xenograft model to further demonstrate the therapeutic potential of systemic delivery of miR-200c to enhance radiosensitivity in lung cancer. Our findings suggest that the antitumor effects of miR-200c result partially from its regulation of the oxidative stress response; they further suggest that miR-200c, in combination with radiation, could represent a therapeutic strategy in the future.

Evaluation of 90-day oral rat toxicity studies on the food additive, gum ghatti.

Gum ghatti, a polysaccharide of natural origin, is used in foods as a thickening, gelling, emulsifying and stabilizing agent. In a 90-day toxicity study following Organization for Economic Co-operation and Development (OECD) Guideline #408, male and female Sprague-Dawley rats were exposed to 0 (control), 0.5, 1.5 and 5% gum ghatti in AIN-93M basal diet. Expected changes included increased full and empty cecal weights in 5% groups. Incidentally 2/10 females from the 5% gum ghatti group had a single colon ulcer with associated acute inflammation. In a second 90-day study increased cecal weights were present in Sprague-Dawley females exposed to 5% gum ghatti in AIN-93M and NIH-07 basal diets. A single colon ulcer with associated acute inflammation occurred in 1/20 control females given AIN-93M basal diet. The colon ulcers were considered a sporadic change possibly attributable to AIN-93M basal diet. In the second study a few statistically significant alterations in clinical chemistry were considered sporadic and unrelated to treatment. Feed consumption among treated and control groups was similar for each sex. Gum ghatti intake at the 5% dietary level ranged from 3044 to 3825mg/kg body weight/day. The 5% dietary administration was a NOAEL in both studies. NOAELs for males and females in the first study were 3044 and 3309mg/kg/day, respectively. NOAELs for females in the second study were 3670 and 3825mg/kg/day for AIN-93M and NIH-07 diets, respectively.

Superoxide dismutase expression and oxidative damage in a case of myopathy in brown pelicans (Pelecanus occidentalis).

Four brown pelicans (Pelecanus occidentalis) housed at a rehabilitation facility were found dead after a 3-day history of muscle weakness and after being fed for about 2 weeks from a recent shipment of fish. The birds had pale streaking of the skeletal and heart muscles. Microscopically, the skeletal muscle, and to a lesser extent the cardiac muscle, had severe myocyte degeneration and necrosis characterized by microvacuolation with loss of cross-striations, condensation of cytoplasm, fragmentation, mineralization, and inflammatory cell infiltrates consisting of multinucleated cells, macrophages, and few heterophils. The findings were consistent with myopathy, and a nutritional myopathy caused by eating rancid fish was suspected. Immunohistochemical staining revealed abundant immunoreactive copper zinc superoxide dismutase and manganese superoxide dismutase either as diffuse homogeneous precipitates or granular aggregates in the cytoplasm of affected cells. Immunoreactivity was directly related to degree of cellular damage as estimated by light microscopic examination. We suggest that the lack of protection, despite upregulation of superoxide dismutase, is most likely attributable to supersaturation of oxidants beyond the capacity of superoxide dismutases to scavenge.

Endosomal transport of ErbB-2: mechanism for nuclear entry of the cell surface receptor.

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 beta1, nuclear pore protein Nup358, and a host of players in endocytic internalization. Knocking down importin beta1 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 beta1 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 beta1 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.

Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2.

Pathological expression of human ErbB-2 protein, also known as HER-2, is common in many types of cancer. ErbB-2 is a member of the EGF receptor tyrosine kinase family and has been rigorously studied as a signaling molecule on the cell membrane. Here, we report that ErbB-2 is also expressed in the nucleus in cultured cells as well as primary tumor tissues. Nuclear ErbB-2 was found to associate with multiple genomic targets in vivo, including the cyclooxygenase enzyme COX-2 gene promoter. ErbB-2 forms a complex at a specific nucleotide sequence of the COX-2 promoter and is able to stimulate its transcription. This study demonstrates the presence of ErbB-2 in the nucleus and identifies the function of ErbB-2 as a transcriptional regulator.