MEK Guards Proteome Stability and Inhibits Tumor-Suppressive Amyloidogenesis via HSF1

SummarySignaling through RAS/MAP kinase pathway is central to biology. ERK has long been perceived as the only substrate for MEK. Here, we report that HSF1, the master regulator of the proteotoxic stress response, is a new MEK substrate. Beyond mediating cell-environment interactions, the MEK-HSF1 regulation impacts malignancy. In tumor cells, MEK blockade inactivates HSF1 and thereby provokes proteomic chaos, presented as protein destabilization, aggregation, and, strikingly, amyloidogenesis. Unlike their non-transformed counterparts, tumor cells are particularly susceptible to proteomic perturbation and amyloid induction. Amyloidogenesis is tumor suppressive, reducing in vivo melanoma growth and contributing to the potent anti-neoplastic effects of proteotoxic stressors. Our findings unveil a key biological function of the oncogenic RAS-MEK signaling in guarding proteostasis and suppressing amyloidogenesis. Thus, proteomic instability is an intrinsic feature of malignant state, and disrupting the fragile tumor proteostasis to promote amyloidogenesis may be a feasible therapeutic strategy

Loss of TRP53 (p53) accelerates tumorigenesis and changes the tumor spectrum of SJL/J mice.

Known as the guardian of the genome, transformation-related protein 53 (TRP53) is a well -known tumor suppressor. Here, we describe a novel TRP53 deficient mouse model on a tumor prone background-SJL/J mice. The absence of TRP53 (TRP53 nullizygosity) leads to a shift in the tumor spectrum from a non-Hodgkin\u27s-like disease to thymic lymphomas and testicular teratomas at a very rapid tumor onset averaging ~12 weeks of age. In haplotype studies, comparing tumor prone versus tumor resistant Trp53 null mouse strains, we found that other tumor suppressor, DNA repair and/or immune system genes modulate tumor incidence in TRP53 null strains, suggesting that even a strong tumor suppressor such as TRP53 is modulated by genetic background. Due to their rapid development of tumors, the SJL/J TRP53 null mice generated here can be used as an efficient chemotherapy or immunotherapy screening mouse model

Evaluation of tarsal injuries in C57BL/6J male mice.

Tarsal joint abnormalities have been observed in aged male mice on a C57BL background. This joint disease consists of calcaneal displacement, inflammation, and proliferation of car- tilage and connective tissue, that can progress to ankylosis of the joint. While tarsal pathol- ogy has been described previously in C57BL/6N substrains, as well as in STR/ort and B10. BR strain, no current literature describes this disease occurring in C57BL/6J mice. More importantly the behavioral features that may result from such a change to the joint have yet to be evaluated. This condition was observed in older male mice of the C57BL/6J lineage, around the age of 20 weeks or older, at a frequency of 1% of the population. To assess potential phenotypic sequela, this study sought to evaluate body weight, frailty assessment, home cage wheel running, dynamic weight bearing, and mechanical allodynia with and with- out the presence of pain relief with morphine. Overall mice with tarsal injuries had signifi- cantly higher frailty scores (p\u3c 0.05) and weighed less (p\u3c0.01) compared to unaffected mice. Affected mice had greater overall touch sensitivity (p\u3c0.05) and they placed more weight on their forelimbs (p\u3c0.01) compared to their hind limbs. Lastly, when housed with a running wheel, affected mice ran for a shorter length of time (p\u3c0.01) but tended to run a greater distance within the time they did run (p\u3c0.01) compared to unaffected mice. When tested just after being given morphine, the affected mice performed more similarly to unaf- fected mice, suggesting there is a pain sensation to this disease process. This highlights the importance of further characterizing inbred mouse mutations, as they may impact research programs or specific study goals

The occurrence of tarsal injuries in male mice of C57BL/6N substrains in multiple international mouse facilities.

Dislocation in hindlimb tarsals are being observed at a low, but persistent frequency in group-housed adult male mice from C57BL/6N substrains. Clinical signs included a sudden onset of mild to severe unilateral or bilateral tarsal abduction, swelling, abnormal hindlimb morphology and lameness. Contraction of digits and gait abnormalities were noted in multiple cases. Radiographical and histological examination revealed caudal dislocation of the calcaneus and partial dislocation of the calcaneoquartal (calcaneus-tarsal bone IV) joint. The detection, frequency, and cause of this pathology in five large mouse production and phenotyping centres (MRC Harwell, UK; The Jackson Laboratory, USA; The Centre for Phenogenomics, Canada; German Mouse Clinic, Germany; Baylor College of Medicine, USA) are discussed

Treatment of murine tumors with recombinant Myxoma viruses

Oncolytic viruses are designed to be novel cancer treatments that not only specifically target cancer cells, but can act as vectors to express proteins of interest within the tumor bed. Myxoma virus (MYXV), a rabbit-specific poxvirus, was evaluated as an oncolytic viral treatment for cancer in immunocompetent mouse models of cancer. In addition, a recombinant MYXV that expresses the murine gene for interleukin 15 (IL-15) was also used, in order to determine if increased expression of IL-15 within the tumor bed could improve MYXV treatment by recruiting additional anti-tumor immune cells to the tumor. In vitro experiments evaluated MYXV infectivity in murine melanoma, glioma, and lymphoma cells using a recombinant MYXV that expresses the fluorescent protein tdTomato (MYXV:Tomato). These studies indicated that B16-F10 melanoma cells were fully-permissive to MYXV infection, while GL261 glioma cells were partially permissive, and EL4 cells were non-permissive. All stages of virus morphology were observed in the permissive cells and there was evidence of cell-to-cell spread of MYXV. Other studies have shown that the susceptibility of human cancer cell lines to MYXV infection depends on the presence of phosphorylated Akt, which was present in the B16-F10 melanoma and GL261 glioma cell lines. Cancer cell death occurred following infection with MYXV and was likely due to cell lysis and necrosis as apoptosis was not evident at 48 hours post infection. Initial in vivo studies using immunocompetent mice bearing intracranial melanoma or lymphoma tumors demonstrated that a single inoculation of intratumoral (IT) MYXV:Tomato was safe but did not produce any significant increase in survival, while studies using intracranial GL261 were inconclusive. However, other studies have shown that MYXV treatment of melanoma not located in the brain can increase survival if multiple injections are given. Therefore, weekly IT injections of MYXV or MYXV expressing the gene for murine IL-15 (MYXV:IL15) were administered in an immunocompetent mouse model of subcutaneous melanoma. The injections were safe and MYXV:IL15 did not cause any detectable adverse clinical signs or any significant increase in viral clearance. A statistically significant increase in survival time was obtained in mice given repeated IT injections of MYXV or MYXV:IL15 as compared to controls. The survival time in tumor-bearing mice treated with MYXV expressing IL-15 was not significantly different than mice treated with MYXV. In addition, MYXV-driven expression of IL-15 was evaluated to determine if it would recruit natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) to the tumor. An extensive pathologic study of the mouse model including histopathology and immunohistochemistry (IHC) was performed at various time points to determine the levels of immune cell infiltration in the tumor and the effects of intratumoral virus inoculation. The number of lymphocytes and neutrophils observed histologically to be infiltrating into the tumors as well as into the tissue surrounding the tumors was significantly increased in mice treated with live MYXV or MYXV:IL15 versus PBS treated control mice. However, IHC revealed that a statistically significant increase in the number of infiltrating cytotoxic lymphocytes (NK cells or CTLs) was not present in MYXV:IL15 treated mice versus control mice, although increased levels of IL-15 were detected within the tumors. Serum neutralization assay revealed that some of the mice treated with MYXV, MYXV:IL15, or UV-MYXV, developed an antibody response to MYXV. The presence of an antibody response did not correlate with survival time or the amount of leukocyte response observed by histology or IHC. In conclusion, treatment with weekly IT injections of MYXV or MYXV:IL15 significantly prolonged survival in mice bearing B16-F10 tumors, which correlated with an increase in the number of neutrophils and lymphocytes infiltrating in and around the tumors, but MYXV:IL15 did not increase survival over MYXV alone

Rapamycin Ameliorates Nephropathy despite Elevating Hyperglycemia in a Polygenic Mouse Model of Type 2 Diabetes, NONcNZO10/LtJ.

While rapamycin treatment has been reported to have a putatively negative effect on glucose homeostasis in mammals, it has not been tested in polygenic models of type 2 diabetes. One such mouse model, NONcNZO10/LtJ, was treated chronically with rapamycin (14 ppm encapsulated in diet) and monitored for the development of diabetes. As expected, rapamycin treatment accelerated the onset and severity of hyperglycemia. However, development of nephropathy was ameliorated, as both glomerulonephritis and IgG deposition in the subendothelial tuft were markedly reduced. Insulin production and secretion appeared to be inhibited, suppressing the developing hyperinsulinemia present in untreated controls. Rapamycin treatment also reduced body weight gain. Thus, rapamycin reduced some of the complications of diabetes despite elevating hyperglycemia. These results suggest that multiple factors must be evaluated when assessing the benefit vs. hazard of rapamycin treatment in patients that have overt, or are at risk for, type 2 diabetes. Testing of rapamycin in combination with insulin sensitizers is warranted, as such compounds may ameliorate the putative negative effects of rapamycin in the type 2 diabetes environment. PLoS One 2014 Dec 4; 9(12):e114324

Effect of rapamycin on kidney glomerular morphology in NONcNZO10 mice.

<p>Untreated mice (A) show hyaline deposits, likely subendothelial, in the glomerular tuft. These deposits are greatly reduced in rapa-treated mice (B). The deposits are strongly positive for IgG in untreated mice (C), and IgG staining is greatly reduced in rapa-treated mice (D). 100 glomeruli from each mouse were scored for presence of nephritis (E) and/or hyaline thrombi deposits (F), showing quantitatively the reduction in kidney disease by rapa treatment.</p

Comparison of NONcNZO10 mice on rapamycin and control diets.

<p>Rapa treatment suppresses body weight gain, elevates plasma glucose, and reduces plasma insulin. All 20 mice were fed 11% fat diet until 12 weeks of age, when 10 were switched to an 11% fat diet containing 14 ppm encapsulated rapamycin. (A) Body weight. (B) Plasma glucose. (C) Plasma insulin. Values ± standard error are shown from 8 to 24 weeks of age.</p

Effect of rapamycin on pancreatic islet morphology in NONcNZO10 mice.

<p>(A) Islet numbers and size variability do not differ between untreated and rapa-treated groups. (B, C) Islets in untreated mice are mostly well granulated, but roughly 38% of islets show some degranulation. (B, D) Islets in rapa-treated mice show more extensive degranulation and some fibrous replacement with occasional fibrous encapsulation. Sections were stained with aldehyde fuchsin.</p

Rapamycin/metformin co-treatment normalizes insulin sensitivity and reduces complications of metabolic syndrome in type 2 diabetic mice

Rapamycin treatment has positive and negative effects on progression of type 2 diabetes (T2D) in a recombinant inbred polygenic mouse model, male NONcNZO10/LtJ (NcZ10). Here, we show that combination treatment with metformin ameliorates negative effects of rapamycin while maintaining its benefits. From 12 to 30 weeks of age, NcZ10 males were fed a control diet or diets supplemented with rapamycin, metformin, or a combination of both. Rapamycin alone reduced weight gain, adiposity, HOMA-IR, and inflammation, and prevented hyperinsulinemia and pre-steatotic hepatic lipidosis, but exacerbated hyperglycemia, hypertriglyceridemia, and pancreatic islet degranulation. Metformin alone reduced hyperinsulinemia and circulating c-reactive protein, but exacerbated nephropathy. Combination treatment retained the benefits of both while preventing many of the deleterious effects. Importantly, the combination treatment reversed effects of rapamycin on markers of hepatic insulin resistance and normalized systemic insulin sensitivity in this inherently insulin-resistant model. In adipose tissue, rapamycin attenuated the expression of genes associated with adipose tissue expansion (Mest, Gpam), inflammation (Itgam, Itgax, Hmox1, Lbp), and cell senescence (Serpine1). In liver, the addition of metformin counteracted rapamycin-induced alterations of G6pc, Ppara, and Ldlr expressions that promote hyperglycemia and hypertriglyceridemia. Both rapamycin and metformin treatment reduced hepatic Fasn expression, potentially preventing lipidosis. These results delineate a state of insulin signaling restriction that withdraws endocrine support for further adipogenesis, progression of the metabolic syndrome, and the development of its comorbidities. Our results are relevant for the treatment of T2D, the optimization of current rapamycin-based treatments for posttransplant rejection and various cancers, and for the development of treatments for healthy aging