Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.
Quiescence is a state of reversible cell cycle arrest that can grant protection against many environmental insults. In some systems, cellular quiescence is associated with a low metabolic state characterized by a decrease in glucose uptake and glycolysis, reduced translation rates and activation of autophagy as a means to provide nutrients for survival. For cells in multiple different quiescence model systems, including Saccharomyces cerevisiae, mammalian lymphocytes and hematopoietic stem cells, the PI3Kinase/TOR signaling pathway helps to integrate information about nutrient availability with cell growth rates. Quiescence signals often inactivate the TOR kinase, resulting in reduced cell growth and biosynthesis. However, quiescence is not always associated with reduced metabolism; it is also possible to achieve a state of cellular quiescence in which glucose uptake, glycolysis and flux through central carbon metabolism are not reduced. In this review, we compare and contrast the metabolic changes that occur with quiescence in different model systems.
NADPH is a critical metabolite that is important for regenerating reduced glutathione from oxidized glutathione and eliminating reactive oxygen species (ROS). Metabolic flux and microarray experiments in fibroblasts demonstrated that NADPH‐producing enzymes, glucose‐6‐phosphate dehydrogenase (G6PD), isocitrate dehydrogenase (IDH), and the upstream transcription factor NRF2, are all activated in quiescent compared with proliferating fibroblasts. We demonstrated that G6PD and NRF2 are functionally important for quiescence by showing that inhibition of G6PD or NRF2 results in oxidative stress and apoptosis specifically in quiescent fibroblasts. Flow cytometry experiments demonstrated that ROS in quiescent cells can be derived from mitochondrial superoxide that results from increased mitochondrial activity in the serum‐starved compared with proliferating fibroblasts, as well as an increase in reactive nitrogen species that arise from peroxisomes. To understand the physiological role of NADPH‐production pathways, we examined their potential activity in mouse skin. Consistent with our findings in quiescent fibroblasts, in situ metabolic activity assays revealed higher potential activity for G6PD and IDH in non‐dividing cells. Of particular interest was the high IDH potential in quiescent hair follicle stem cells. Staining of live mouse skin with monochlorobimane showed higher reactivity, consistent with higher levels of reduced glutathione, in hair follicle stem cells. Inhibition of IDH activity in healthy mouse skin with two different inhibitors resulted in progression in the hair follicle cycle from a quiescent to proliferative state suggesting that the high IDH activity observed in hair follicle stem cells may contribute to the maintenance of these stem cells in a quiescent state. Going forward, our focus is on understanding the role of IDH in the maintenance of stem cells as opposed to proliferating, committed progenitor cells. Support or Funding Information H.A.C. was the Milton E. Cassel scholar of the Rita Allen Foundation. This work was supported by NIGMS Center of Excellence grant P50 GM071508, two grants from the Cancer Institute of New Jersey, the New Jersey Commission on Cancer Research, National Cancer Institute 1RC1 CA147961‐01, a Focused Funding Grant to H.A.C. from the Johnson & Johnson Foundation and a grant from the PhRMA Foundation to H.A.C, and XXXX to W.E.L. J.M.S. was supported by NIH training grant T32 HG003284. E.M.H. acknowledges a Bowen Fellowship from Princeton University and the New Jersey Commission on Cancer Research. E.J.S. acknowledges support from the National Science Foundation.
Abstract Objectives: The aim of this study is to investigate quiescence-related resistance to proteasome-inhibition mediated proteotoxicity, and its regulatory mechanisms. Introduction: Quiescence, the reversible arrest of cellular proliferation, is critical for normal cellular development and tissue maintenance. Moreover, while proteasome inhibitors (PIs) are used to treat specific cancers, it has been shown that subsets of cells within a tumor, through quiescence, avoid PI-induced apoptosis and survive. Thus, deciphering the mechanisms through which quiescent cells resist proteasome inhibition is of basic and clinical relevance. Methods: Proliferating and quiescent primary human fibroblasts were used as a model system to study the effects of PIs (MG132, epoxomycin and Bortezomib). Flow cytometry, Western blots, microscopy and microarrays were used to identify specific pathways activated by proliferating and quiescent fibroblasts in response to proteasome inhibition. Results: We found that the proteasome-inhibited quiescent fibroblasts maintain viability while proliferating fibroblasts rapidly undergo apoptosis. We further found that proteasome-inhibited proliferating and quiescent fibroblasts up-regulate multiple protective pathways, including authophagy, chaperones and antioxidants, and evidence no observable difference in proteasome-mediated degradation pathway activity. However, we found that in proliferating fibroblasts, proteasome inhibition leads to increased reactive oxygen species (ROS), peri-nuclear protein aggresomes, and apoptotic cell death. By comparison, proteasome-inhibited quiescent cells display selective up-regulation of MnSOD (a ROS detoxifying enzyme), dispersed protein aggregates, and less apoptotic cell death. Knockdown of p62/SQSTM1 (a protein important for aggresome formation) or treatment with an antioxidant N-acetylcysteine results in reduced aggresome formation and decreased apoptosis in response to proteasome inhibition. Moreover, inhibition of autophagy sensitizes serum-starved quiescent cells to proteasome inhibition induced apoptosis. This suggests that multiple mechanisms, including autophagy, ROS detoxification and differential subcellular trafficking of ubiquitinated proteins are implicated in quiescence-related resistance to PI-induced apoptosis. These results point to possible strategies for improving the effectiveness of PIs in cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2033. doi:1538-7445.AM2012-2033
Proteasome inhibition is used as a treatment strategy for multiple types of cancers. Although proteasome inhibition can induce apoptotic cell death in actively proliferating cells, it is less effective in quiescent cells. In this study, we used primary human fibroblasts as a model system to explore the link between the proliferative state of a cell and proteasome inhibition-mediated cell death. We found that proliferating and quiescent fibroblasts have strikingly different responses to MG132, a proteasome inhibitor; proliferating cells rapidly apoptosed, whereas quiescent cells maintained viability. Moreover, MG132 treatment of proliferating fibroblasts led to increased superoxide anion levels, juxtanuclear accumulation of ubiquitin- and p62/SQSTM1-positive protein aggregates, and apoptotic cell death, whereas MG132-treated quiescent cells displayed fewer juxtanuclear protein aggregates, less apoptosis, and higher levels of mitochondrial superoxide dismutase. In both cell states, reducing reactive oxygen species with N-acetylcysteine lessened protein aggregation and decreased apoptosis, suggesting that protein aggregation promotes apoptosis. In contrast, increasing cellular superoxide levels with 2-methoxyestradiol treatment or inhibition of autophagy/lysosomal pathways with bafilomycin A1 sensitized serum-starved quiescent cells to MG132-induced apoptosis. Thus, antioxidant defenses and the autophagy/lysosomal pathway protect serum-starved quiescent fibroblasts from proteasome inhibition-induced cytotoxicity.
A balance between angiogenesis inducers and inhibitors in the microenvironment controls the rate of new blood vessel formation. We hypothesized that fibroblasts, an important cellular constituent of the tissue stroma, secrete molecules that contribute to this balance. We further hypothesized that fibroblasts secrete molecules that promote angiogenesis when they are in a proliferative state and molecules that inhibit angiogenesis when they are not actively cycling (quiescent). Microarray analysis revealed that angiogenesis inducers and inhibitors are regulated as fibroblasts transition into a quiescent state and re-enter the cell cycle in response to changes in serum. To assess whether changes in transcript levels result in changes in the levels of secreted proteins, we collected conditioned medium from proliferating and quiescent fibroblasts and performed immunoblotting for selected proteins. Secreted protein levels of the angiogenesis inhibitor PEDF were higher in quiescent than proliferating fibroblasts. Conversely, proliferating fibroblasts secreted increased levels of the angiogenesis inducer VEGF-C. For the angiogenesis inhibitor thrombospondin-2, quiescent cells secreted a prominent 160 kDa form in addition to the 200 kDa form secreted by proliferating and restimulated fibroblasts. Using immunohistochemistry we discovered that fibroblasts surround blood vessels and that the angiogenesis inhibitor PEDF is expressed by quiescent fibroblasts in uterine tissue, supporting a role for PEDF in maintaining quiescence of the vasculature. This work takes a new approach to the study of angiogenesis by examining the expression of multiple angiogenesis genes secreted from a key stromal cell, the fibroblast.
Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.
