Both targeted inhibition of oncogenic driver mutations and immune-based therapies show efficacy in treatment of patients with metastatic cancer but responses can be either short-lived or incompletely ...effective. Oncogene inhibition can augment the efficacy of immune-based therapy but mechanisms by which these two interventions might cooperate are incompletely resolved. Using a novel transplantable BRAF
V600E
-mutant murine melanoma model (SB-3123), we explore potential mechanisms of synergy between the selective BRAF
V600E
inhibitor vemurafenib and adoptive cell transfer (ACT)-based immunotherapy. We found that vemurafenib cooperated with ACT to delay melanoma progression without significantly affecting tumor infiltration or effector function of endogenous or adoptively transferred CD8
+
T cells as previously observed. Instead, we found that the T-cell cytokines IFNγ and TNFα synergized with vemurafenib to induce cell-cycle arrest of tumor cells
in vitro
. This combinatorial effect was recapitulated in human melanoma-derived cell lines and was restricted to cancers bearing a BRAF
V600E
-mutation. Molecular profiling of treated SB-3123 indicated that the provision of vemurafenib promoted the sensitization of SB-3123 to the anti-proliferative effects of T-cell effector cytokines. The unexpected finding that immune cytokines synergize with oncogene inhibitors to induce growth arrest have major implications for understanding cancer biology at the intersection of oncogenic and immune signaling and provides a basis for design of combinatorial therapeutic approaches for patients with metastatic cancer.
Although many melanomas harbor either activating mutations in BRAF or NRAS, there remains a substantial group of tumors without either mutation about which little is known. Here, we used a genomic ...strategy to define a novel group of melanoma cell lines with co-overexpression of CDK4 and KIT. Although this sub-group lacked any known KIT mutations, they had high phospho-KIT receptor expression - indicating receptor activity. fuantitative PCR confirmed the existence of a similar KIT/CDK4 sub-group in human melanoma samples. Pharmacological studies showed the KIT/CDK4 overexpressing sub-group to be resistant to BRAF inhibitors but sensitive to imatinib in both
in vitro
and
in vivo
melanoma models. Mechanistically, imatinib treatment led to increased apoptosis and G1-phase cell cycle arrest associated with the inhibition of phospho-ERK and increased expression of p27
KIP
. Other melanoma cell lines, which retained some KIT expression but lacked phospho-KIT, were not sensitive to imatinib, suggesting that KIT expression alone is not predictive of response. We suggest that co-overexpression of KIT/CDK4 is a potential mechanism of oncogenic transformation in some BRAF/NRAS wild-type melanomas. This group of melanomas may be a sub-population for which imatinib or other KIT inhibitors may constitute optimal therapy.
The purpose of this study was to develop a method to highlight immune cells and cell clusters in histology tissue stained with single or multiple biomarkers. B6 mice were implanted (rear flank) with ...YUMM/YUMMER1.7 melanoma cells and treated with or without immunotherapy (PD-1, CTLA-4). Skin, lymph nodes, and spleens were formalin-fixed paraffin-embedded and stained by immunohistochemistry (IHC) or immunofluorescence (IF). Biomarkers for B cells (CD20), neutrophils (Ly6B.2), melanoma cells (GFP), helper T cells (CD4), and helper T cells activity (Granzyme B, IL-6, Ki-67, T-bet, Foxp3, RORu03b3(t)) were combined in single or multiple staining rounds. The TissueFAXS (TissueGnostics, Vienna, Austria) imaging system was used to acquire image tiles for analysis. Image processing and analysis was performed using StrataQuest software (TissueGnostics). An image stitching algorithm was used on the tiled images to reconstruct the whole image. Algorithms were created to align the stitched images, generate a composite image consisting of all biomarkers, isolate each cell in the composite image, and identify the positive cells in the composite image. Tissue cytometry coupled with backgating into the tissue images was used to visualize, quantitate, and validate the data. The results for IHC showed that B cell and neutrophil density was significantly increased during the natural host response in the time period post melanoma cell implantation corresponding to an adaptive immune response (days 7-19) versus immunosurveillance (days 0u20136) (p<0.01, p<0.001). The density was further increased with immunotherapy treatment (p<0.01, p<0.05). For IF, using in silico analysis, we could identify three distinct clusters of CD4 expressing cell populations: intermediate, high, and mixed. Each CD4 cell cluster was composed of different combinations of biomarkers of CD4 activity. In summary, we developed a powerful method that utilized tissue cytometry to highlighting immune cells and immune cell clusters.
Membrane-anchored transforming growth factor alpha (proTGF alpha) belongs to a group of transmembrane proteins whose extracellular domains are selectively cleaved and released into the medium. We ...demonstrate that the carboxy-terminal valine in the cytoplasmic tail of proTGF alpha is required for cleavage of the growth factor ectodomain in response to various activators. This cleavage process occurs outside Golgi or lysosomal locations, affects cell surface proTGF alpha, and requires little or no membrane traffic. We propose that cleavage and release of proTGF alpha ectodomain involve a specialized proteolytic system and depend on the recognition of a simple and specific determinant located in the proTGF alpha cytoplasmic tail.
Summary
There has been uncertainty regarding the precise role that the pocket protein Rb1 plays in murine melanocyte homeostasis. It has been reported that the TAT‐Cre mediated loss of exon 19 from a ...floxed Rb1 allele causes melanocyte apoptosis in vivo and in vitro. This is at variance with other findings showing, either directly or indirectly, that Rb1 loss in melanocytes has no noticeable effect in vivo, but in vitro leads to a semi‐transformed phenotype. In this study, we show that Rb1‐null melanocytes lacking exon 19 do not undergo apoptosis and survive both in vitro and in vivo, irrespective of the developmental stage at which Cre‐mediated ablation of the exon occurs. Further, Rb1 loss has no serious long‐term ramifications on melanocyte homeostasis in vivo, with Rb1‐null melanocytes being detected in the skin after numerous hair cycles, inferring that the melanocyte stem cell population carrying the Cre‐mediated deletion is maintained. Consequently, whilst Rb1 loss in the melanocyte is able to alter cellular behaviour in vitro, it appears inconsequential with respect to melanocyte homeostasis in the mouse skin.
Mutational activation of BRAF is the earliest and most common genetic alteration in human melanoma. To build a model of human melanoma, we generated mice with conditional melanocyte-specific ...expression of Braf.sup.V600E. Upon induction of Braf.sup.V600E expression, mice developed benign melanocytic hyperplasias that failed to progress to melanoma over 15-20 months. By contrast, expression of Braf.sup.V600E combined with Pten tumor suppressor gene silencing elicited development of melanoma with 100% penetrance, short latency and with metastases observed in lymph nodes and lungs. Melanoma was prevented by inhibitors of mTorc1 (rapamycin) or MEK1/2 (PD325901) but, upon cessation of drug administration, mice developed melanoma, indicating the presence of long-lived melanoma-initiating cells in this system. Notably, combined treatment with rapamycin and PD325901 led to shrinkage of established melanomas. These mice, engineered with a common genetic profile to human melanoma, provide a system to study melanoma's cardinal feature of metastasis and for preclinical evaluation of agents designed to prevent or treat metastatic disease.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The membrane-anchored forms of transforming growth factor-alpha (TGF-alpha) and stem cell growth factors (Kit ligands) KL-1
and KL-2 are converted to soluble growth factor forms by a regulated ...proteolytic cleavage process. Each of these proteins
is cleaved at a distinct site, however their cleavage is activated via a common set of intracellular signaling mechanisms.
By using a panel of protease inhibitors, we show here that at least two cell-associated serine protease activities with distinct
specificities participate in membrane growth factor cleavage. Two serine protease inhibitors of broad specificity, diisopropylfluorophosphate
and 3,4-dichloroisocoumarin, prevent the cleavage of proTGF-alpha and KL-1 but not that of KL-2. Of the agents tested, N-tosyl-L-phenylalanine
chloromethyl ketone and various haloenol lactone derivatives are the most potent inhibitors of cleavage of all three membrane
growth factors. It is concluded that cleavage of membrane-anchored growth factors involves a proteolytic system with multiple
serine protease activities regulated through common mechanisms.
The telomere-stabilizing enzyme telomerase is induced in tumors and functionally associated with unlimited replicative potential. To further explore its necessity, transgenic mice expressing SV40 or ...HPV16 oncogenes, which elicit carcinomas in pancreas and skin, respectively, were rendered telomerase-deficient. Absence of telomerase had minimal impact on tumorigenesis, even in
terc
−/−
generations (G5–7) exhibiting shortened telomeres and phenotypic abnormalities in multiple organs. Analyses of chromosomal aberrations were not indicative of telomere dysfunction or increased genomic instability in tumors. Quantitative image analysis of telomere repeat intensities comparing biopsies of skin hyperplasia, dysplasia, and carcinoma revealed that telomere numbers and relative lengths were maintained during progression, implicating a means for preserving telomere repeats and functionality in the absence of telomerase.
PDF
