Autophagy In Cancer-associated Fibroblasts Research Articles

Abstract 6803: TRIM16 regulates IL-6 secretion in head and neck cancer-associated fibroblasts

Abstract Despite current therapy, head and neck squamous cell carcinoma (HNSCC), the 7th most common cancer worldwide, is associated with high morbidity and mortality. Response to current therapies could be improved with a better understanding of the tumor microenvironment of HNSCC. We previously reported that cancer-associated fibroblasts (CAFs), the most abundant stromal cell type in the HNSCC microenvironment, have elevated levels of basal autophagy than oral fibroblasts from cancer-free subjects (NF). Disrupting autophagy in CAFs with Beclin-1 siRNA reduced secretion of IL-6, IL-8, and other factors known to promote HNSCC progression. The recent emerging role of autophagy in secretion of tumor promoting factors has high impact because (i) the mechanism can provide a new target to block the secretion of protumor factors from CAFs that contribute to HNSCC progression, and (ii) these cytokines can serve as potential indicators for the efficacy of autophagy-targeted therapies. To this end, we carried out an unbiased assessment of the proteins associated with autophagosomes in primary CAFs from HNSCC samples using mass spectrometry. We performed immunoprecipitation of LC3B from the isolated small vesicles of the CAFs, identified and validated the association of several trafficking proteins in secretory autophagy including VAMP3 and SNAP23. Further, the tripartite motif (TRIM) proteins have been shown to mediate the fate of autophagosomes either for degradation or secretion. We hypothesized that TRIM proteins are involved in transport of secretory autophagosomes. Our data demonstrate CAFs express higher levels of TRIM16 mRNA than NF. We demonstrate immunogold labeling of TRIM16 or IL-6 localized to the autophagosomes using transmission electron microscopy. Further, using immunofluorescence, we demonstrated the colocalization of TRIM16 and IL-6, as well as IL-6 and LC3B in cytoplasmic puncta. In addition, we used proximity ligation assays to confirm the interaction of TRIM16 and IL-6, as well as IL-6 and LC3B. TRIM16 knockdown using siRNA in CAFs reduced the secretion of IL-6 as measured by ELISA. In conclusion, we demonstrate an important role of TRIM16 in secretory autophagy making it a potential therapeutic target to mitigate CAF-mediated HNSCC growth. Citation Format: Thuc Ly, Bailey Pickard, Avisha Pandey, Noraida Martinez-Rivera, Eduardo Rosa-Molinar, Michael Washburn, Wen-Xing Ding, Sufi Mary Thomas. TRIM16 regulates IL-6 secretion in head and neck cancer-associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6803.

TCF12 regulates exosome release from epirubicin-treated CAFs to promote ER+ breast cancer cell chemoresistance

Cancer-associated fibroblasts (CAFs) are the predominant stromal cells in the microenvironment and play important roles in tumor progression, including chemoresistance. However, the response of CAFs to chemotherapeutics and their effects on chemotherapeutic outcomes are largely unknown. In this study, we showed that epirubicin (EPI) treatment triggered ROS which initiated autophagy in CAFs, TCF12 inhibited autophagy flux and further promoted exosome secretion. Inhibition of EPI-induced reactive oxygen species (ROS) production with N-acetyl-L-cysteine (NAC) or suppression of autophagic initiation with short interfering RNA (siRNA) against ATG5 blunted exosome release from CAFs. Furthermore, exosome secreted from EPI-treated CAFs not only prevented ROS accumulation in CAFs but also upregulated the CXCR4 and c-Myc protein levels in recipient ER+ breast cancer cells, thus promoting EPI resistance of tumor cells. Together, the current study provides novel insights into the role of stressed CAFs in promoting tumor chemoresistance and reveal a new function of TCF12 in regulating autophagy impairment and exosome release.

Cancer-associated fibroblasts promote malignant phenotypes of prostate cancer cells via autophagy

Dysregulation of autophagy in cancer-associated fibroblasts (CAFs) has been demonstrated to play a role in malignant phenotypes of human tumors. We intended to investigate the function of CAFs autophagy in prostate cancer (PCa). Firstly, CAFs and normal fibroblasts (NFs) were isolated from cancerous and adjacent normal tissues of PCa patients, for the following experimental preparation. In comparison with NFs, CAFs expressed higher levels of the myofibroblast marker ?-smooth muscle actin (?-SMA) and the mesenchymal marker Vimentin. Besides, CAFs possessed a higher autophagic level than NFs. As for malignant phenotypes, PCa cells co-cultured with CAFs-CM showed greater proliferation, migration and invasion capabilities, while these outcomes were obviously abolished by autophagy inhibition with 3-Methyladenine (3-MA). Moreover, silencing of ATG5 in CAFs inhibited fibroblasts autophagic level and suppressed malignant phenotypes of PCa cells, while ATG5 overexpression in NFs exerted opposite effects. Depletion of ATG5 in CAFs inhibited the xenograft tumor growth and lung metastasis of PCa cells. Taken together, our data demonstrated the promotive effect of CAFs on PCa malignant phenotypes through ATG5-dependent autophagy, suggesting a novel mechanism for PCa progression.

Tumor Cell Derived Lnc-FSD2-31:1 Contributes to Cancer-Associated Fibroblasts Activation in Pancreatic Ductal Adenocarcinoma Progression through Extracellular Vesicles Cargo MiR-4736.

Pancreatic ductal adenocarcinoma (PDAC) presents with high mortality and short overall survival. Cancer-associated fibroblasts (CAFs) act as refuge for cancer cells in PDAC. Mechanisms of intracelluar communication between CAFs and cancer cells need to be explored. Long noncoding RNAs (lncRNAs) are involved in the modulation of oncogenesis and tumor progression of PDAC; however, specific lncRNAs and their mechanism of action have not been clarified clearly in tumoral microenvironment. This work aims to identify novel lncRNAs involved in cellular interaction between cancer cells and CAFs in PDAC. To this end, differentially expressed lncRNAs between long-term and short-term survival PDAC patients are screened. Lnc-FSD2-31:1 is found to be significantly increased in long-term survival patients. This work then discovers that tumor-derived lnc-FSD2-31:1 restrains CAFs activation via miR-4736 transported by extracellular vesicles (EVs) in vitro and in vivo. Mechanistically, EVs-derived miR-4736 suppresses autophagy and contributes to CAFs activation by targeting ATG7. Furthermore, blocking miR-4736 suppresses tumor growth in genetically engineered KPC (LSL-KrasG12D/+, LSL-Trp53R172H/+, and Pdx-1-Cre) mouse model of PDAC. This study demonstrates that intratumoral lnc-FSD2-31:1 modulates autophagy in CAFs resulting in their activation through EVs-derived miR-4736. Targeting miR-4736 may be a potential biomarker and therapeutic target for PDAC.

TGF-β1 induced autophagy in cancer associated fibroblasts during hypoxia contributes EMT and glycolysis via MCT4 upregulation

The Transforming growth factor-β1 (TGF- β1) in the tumor microenvironment (TME) is the major cytokine that acts as a mediator of tumor-stroma crosstalk, which in fact has a dual role in either promoting or suppressing tumor development. The cancer-associated fibroblasts (CAFs) are the major cell types in the TME, and the interaction with most of the epithelial cancers is the prime reason for cancer survival. However, the molecular mechanisms, associated with the TGF- β1 induced tumor promotion through tumor-CAF crosstalk are not well understood. In the Reverse Warburg effect, CAFs feed the adjacent cancer cells by lactate produced during the aerobic glycolysis. We hypothesized that the monocarboxylate transporter, MCT4 which is implicated in lactate efflux from the CAFs, must be overexpressed in the CAFs. Contextually, to explore the role of TGF- β1 in the hypoxia-induced autophagy in CAFs, we treated CoCl2 and external TGF- β1 to the human dermal fibroblasts and L929 murine fibroblasts. We demonstrated that hypoxia accelerated the TGF- β1 signaling and subsequent transformation of normal fibroblasts to CAFs. Moreover, we elucidated that synergistic induction of autophagy by hypoxia and TGF- β1 upregulate the aerobic glycolysis and MCT4 expression in CAFs. Furthermore, we showed a positive correlation between glucose consumption and MCT4 expression in the CAFs. Autophagy was also found to be involved in the EMT in hypoxic CAFs. Collectively, these findings reveal the unappreciated role of autophagy in TME, which enhances the CAF transformation and that promotes tumor migration and metastasis via the reverse Warburg effect.

TGF-β1 Induced Autophagy in Cancer Associated Fibroblasts During Hypoxia Contributes EMT and Glycolysis Via MCT4 Upregulation

TGF-β1 Induced Autophagy in Cancer Associated Fibroblasts During Hypoxia Contributes EMT and Glycolysis Via MCT4 Upregulation

Cancer-Associated Fibroblasts Regulate Bladder Cancer Invasion and Metabolic Phenotypes through Autophagy.

Recently, both cancer-associated fibroblasts (CAFs) and autophagy have been proven to play an important role in tumor development, including bladder cancer (BCa). However, the real mechanisms remain largely unclear. Here, we reconstruct a mimic tumor microenvironment to explore the interaction between CAFs and the BCa cell line T24 using a coculture system. Autophagy in CAFs was induced or inhibited by rapamycin or siRNA, respectively. After coculture with CAFs, T24 cell proliferation, invasion, and aerobic glycolysis were tested in vitro. Rapamycin induced and siAtg5 inhibited autophagy in CAFs. Enhanced autophagy in CAFs promoted cell proliferation and invasion in T24 cells in vitro, while there was no significant difference between the autophagy-inhibited group and the controls. Lactate concentration was elevated in both rapamycin-treated and siAtg5-treated groups compared with the control group. In addition, the expression levels of MCT1, MCT4, HK2, SLC2A1, and MMP-9 were all increased in T24 cells in the autophagy-enhanced group. Our results indicated that CAFs could regulate BCa invasion and metabolic phenotypes through autophagy, providing us with new alternative treatments for BCa in the future.

Active autophagy in cancer-associated fibroblasts: Recent advances in understanding the novel mechanism of tumor progression and therapeutic response.

Autophagy is primarily a homeostatic and catabolic process that is increasingly being recognized to have a pivotal role in the initiation and maintenance of cancer cells, as well as in the emergence of therapeutic resistance. Moreover, in the tumor microenvironment (TME) autophagy plays a crucial and sometimes dichotomous role in tumor progression. Recent studies show that during the early stages of tumor initiation, autophagy suppresses tumorigenesis. However, in the advanced stage of tumorigenesis, autophagy promotes cancer progression by protecting cancer cells against stressful conditions and therapeutic assault. Specifically, in cancer-associated fibroblasts (CAFs), autophagy promotes tumorigenesis not only by providing nutrients to the cancerous cells but also by inducing epithelial to mesenchymal transition, angiogenesis, stemness, and metastatic dissemination of the cancer cells, whereas in the immune cells, autophagy induces the tumor-localized immune response. In the TME, CAFs play a crucial role in cancer cell metabolism, immunoreaction, and growth. Therefore, targeting autophagy in CAFs by several pharmacological inducers like rapamycin or the inhibitor such as chloroquine has gained importance in preclinical and clinical trials. In the present review, we summarized the basic mechanism of autophagy in CAFs along with its role in driving tumorigenic progression through several emerging as well as classical hallmarks of cancer. We also addressed various autophagy inducers as well as inhibitors of autophagy for more efficient cancer management. Eventually, we prioritized some of the outstanding issues that must be addressed with utmost priority in the future to elucidate the role of autophagy in CAFs on tumor progression and therapeutic intervention.

Autophagy in cancer associated fibroblasts promotes lung adenocarcinoma cells migration and invasion

Objective Autophagy is a process by which cellular material is delivered to lysosomes for degradation, providing energy and macromolecular precursors. Cancer-associated fibroblasts (CAFs) have an important role in reprogramming the tumor microenvironment, and exhibit enhanced migratory capacity through autocrine and paracrine. This study aims to explore the possible effects of CAFs autophagy on lung adenocarcinoma cells migration and invasion. Methods Lung adenocarcinoma cell line A549 was co-cultured with or without CAFs via Transwell and Matrige lassay under the same culture condition, the effects of CAFs autophagy on the migration and invasion of A549 were observed. Results We demonstrate autophagy activated CAFs have great ability than CAFs to enhance the migration [(122.7±17.6) cells/cm2 vs. (269.3±72.7) cells/cm2] and invasion [(124.3±19.6) cells/cm2 vs. (280.0±32.1) cells/cm2] of lung adenocarcinoma (LUAD) cell line (F=18.640, 80.630, P<0.05). Interleukin 6 [(545.5±28.2) ng/L vs. (865.4±70.9) ng/L] and interleukin 8 [(374.2±62.2) ng/L vs. (672.4±24.2) ng/L] were highly expressed in the autophagy activated CAFs conditioned media (t=7.261, 7.738, P<0.05). Conclusion Our results establish an oncogenic function for secretory autophagy in CAFs that promote LUAD migration and invasion. Key words: Lung adenocarcinoma; Cancer-associated fibroblasts; Autophagy; Migration and invasion

Blocking Autophagy in Cancer-Associated Fibroblasts Supports Chemotherapy of Pancreatic Cancer Cells.

In this study we evaluated the interaction of pancreatic cancer cells, cancer-associated fibroblasts, and distinct drugs such as α-cyano-4-hydroxycinnamate, metformin, and gemcitabine. We observed that α-cyano-4-hydroxycinnamate as monotherapy or in combination with metformin could significantly induce collagen I deposition within the stromal reaction. Subsequently, we demonstrated that cancer-associated fibroblasts impaired the anti-proliferation efficacy of α-cyano-4-hydroxycinnamate, metformin and gemcitabine. Interestingly, inhibition of autophagy in these fibroblasts can augment the anti-proliferation effect of these chemotherapeutics in vitro and can reduce the tumor weight in a syngeneic pancreatic cancer model. These results suggest that inhibiting autophagy in cancer-associated fibroblasts may contribute to strategies targeting cancer.

Chloroquine plays a cell-dependent role in the response to treatment of pancreatic adenocarcinoma.

In this study, our aim is to assess the role played by autophagy and its inhibition in the different PDAC cellular compartments, and its involvement in chemo-resistance using primary human pancreatic cancer-derived cells (PCC) and Cancer Associated Fibroblasts (CAF). Autophagy flux, as measured by LC3-I and -II in the presence of Chloroquine, showed a variable level in PCC and CAFs. We found no correlation between autophagy level and degree of tumor differentiation. Association of Chloroquine with gemcitabine, 5FU, oxaliplatin, irinotecan and docetaxel revealed that its effect on survival is cell- and drug-dependent in vitro and in vivo. In addition, we demonstrated that autophagy in CAFs can play an important role in sensitizing PDAC to anticancer treatments since its inhibition increased the resistance of PCCs to gemcitabine. In conclusion, this work clearly shows a heterogeneity in the effect of Chloroquine and highlights a role of CAFs autophagy in sensitizing tumors to treatments. It also reveals that the role of autophagy is more complex than expected in PDAC as well as its sensitivity to treatments.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Secretory Autophagy in Cancer-Associated Fibroblasts Promotes Head and Neck Cancer Progression and Offers a Novel Therapeutic Target.

Despite therapeutic advancements, there has been little change in the survival of patients with head and neck squamous cell carcinoma (HNSCC). Recent results suggest that cancer-associated fibroblasts (CAF) drive progression of this disease. Here, we report that autophagy is upregulated in HNSCC-associated CAFs, where it is responsible for key pathogenic contributions in this disease. Autophagy is fundamentally involved in cell degradation, but there is emerging evidence that suggests it is also important for cellular secretion. Thus, we hypothesized that autophagy-dependent secretion of tumor-promoting factors by HNSCC-associated CAFs may explain their role in malignant development. In support of this hypothesis, we observed a reduction in CAF-facilitated HNSCC progression after blocking CAF autophagy. Studies of cell growth media conditioned after autophagy blockade revealed levels of secreted IL6, IL8, and other cytokines were modulated by autophagy. Notably, when HNSCC cells were cocultured with normal fibroblasts, they upregulated autophagy through IL6, IL8, and basic fibroblast growth factor. In a mouse xenograft model of HNSCC, pharmacologic inhibition of Vps34, a key mediator of autophagy, enhanced the antitumor efficacy of cisplatin. Our results establish an oncogenic function for secretory autophagy in HNSCC stromal cells that promotes malignant progression. Cancer Res; 77(23); 6679-91. ©2017 AACR.

High-mobility group box 1 released by autophagic cancer-associated fibroblasts maintains the stemness of luminal breast cancer cells.

Cancer stem cells/cancer-initiating cells (CICs) and their microenvironmental niche play a vital role in malignant tumour recurrence and metastasis. Cancer-associated fibroblasts (CAFs) are major components of the niche of breast cancer-initiating cells (BCICs), and their interactions may profoundly affect breast cancer progression. Autophagy has been considered to be a critical process for CIC maintenance, but whether it is involved in the cross-talk between CAFs and CICs to affect tumourigenesis and pathological significance has not been determined. In this study, we found that the presence of CAFs containing high levels of microtubule-associated protein 1 light chain 3 (LC3II), a marker of autophagosomes, was associated with more aggressive luminal human breast cancer. CAFs in human luminal breast cancer tissues with high autophagy activity enriched BCICs with increased tumourigenicity. Mechanistically, autophagic CAFs released high-mobility group box 1 (HMGB1), which activated its receptor, Toll-like receptor (TLR) 4, expressed by luminal breast cancer cells, to enhance their stemness and tumourigenicity. Furthermore, immunohistochemistry of 180 luminal breast cancers revealed that high LC3II/TLR4 levels predicted an increased relapse rate and a poorer prognosis. Our findings demonstrate that autophagic CAFs play a critical role in promoting the progression of luminal breast cancer through an HMGB1-TLR4 axis, and that both autophagy in CAFs and TLR4 on breast cancer cells constitute potential therapeutic targets. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

MiR-31 affects colorectal cancer cells by inhibiting autophagy in cancer-associated fibroblasts.

Autophagy is a double-edged sword in tumor development. Recent studies have found that miRNAs have an inhibitory effect on the regulation of autophagy. It has been reported that miR-31 plays an important role in the development of colorectal cancer. However, what role miR-31 plays in colorectal cancer-associated fibroblasts (CAFs) has not been determined. In this study, we confirmed that the expression of miR-31 in CAFs was higher than in normal colorectal fibroblasts (NFs). We also found that treatment of CAFs with miR-31 mimic inhibited the expression of the autophagy-related genes Beclin-1, ATG, DRAM and LC3. In addition, we found up-regulation of miR-31 significantly affected colorectal cancer cell behaviors, including proliferation, invasion and apoptosis. Also, up-regulation of miR-31 in CAF could increase the radiosensitivity of colorectal cancer cells co-cultured with CAF. In summary, miR-31 can inhibit autophagy in colorectal CAFs, affect colorectal cancer development, and increase the radiosensitivity of colorectal cancer cells co-cultured with CAF. We hypothesize that miR-31 may become a new target of treatments for colorectal cancer.

CDK inhibitors (p16/p19/p21) induce senescence and autophagy in cancer-associated fibroblasts, “fueling” tumor growth via paracrine interactions, without an increase in neo-angiogenesis

Here, we investigated the compartment-specific role of cell cycle arrest and senescence in breast cancer tumor growth. For this purpose, we generated a number of hTERT-immortalized senescent fibroblast cell lines overexpressing CDK inhibitors, such as p16(INK4A), p19(ARF) or p21(WAF1/CIP1). Interestingly, all these senescent fibroblast cell lines showed evidence of increased susceptibility toward the induction of autophagy (either at baseline or after starvation), as well as significant mitochondrial dysfunction. Most importantly, these senescent fibroblasts also dramatically promoted tumor growth (up to ~2-fold), without any comparable increases in tumor angiogenesis. Conversely, we generated human breast cancer cells (MDA-MB-231 cells) overexpressing CDK inhibitors, namely p16(INK4A) or p21(WAF1/CIP1). Senescent MDA-MB-231 cells also showed increased expression of markers of cell cycle arrest and autophagy, including β-galactosidase, as predicted. Senescent MDA-MB-231 cells had retarded tumor growth, with up to a near 2-fold reduction in tumor volume. Thus, the effects of CDK inhibitors are compartment-specific and are related to their metabolic effects, which results in the induction of autophagy and mitochondrial dysfunction. Finally, induction of cell cycle arrest with specific inhibitors (PD0332991) or cellular stressors [hydrogen peroxide (H₂O₂) or starvation] indicated that the onset of autophagy and senescence are inextricably linked biological processes. The compartment-specific induction of senescence (and hence autophagy) may be a new therapeutic target that could be exploited for the successful treatment of human breast cancer patients.

Autophagy in cancer associated fibroblasts promotes tumor cell survival

Recently, using a co-culture system, we demonstrated that MCF7 epithelial cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal caveolin-1 (Cav-1). However, the detailed signaling mechanism(s) underlying this process remain largely unknown. Here, we show that hypoxia is sufficient to induce the autophagic degradation of Cav-1 in stromal fibroblasts, which is blocked by the lysosomal inhibitor chloroquine. Concomitant with the hypoxia-induced degradation of Cav-1, we see the upregulation of a number of well-established autophagy/mitophagy markers, namely LC3, ATG16L, BNIP3, BNIP3L, HIF-1α and NFκB. In addition, pharmacological activation of HIF-1α drives Cav-1 degradation, while pharmacological inactivation of HIF-1 prevents the downregulation of Cav-1. Similarly, pharmacological inactivation of NFκB-another inducer of autophagy-prevents Cav-1 degradation. Moreover, treatment with an inhibitor of glutathione synthase, namely BSO, which induces oxidative stress via depletion of the reduced glutathione pool, is sufficient to induce the autophagic degradation of Cav-1. Thus, it appears that oxidative stress mediated induction of HIF1- and NFκB-activation in fibroblasts drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we show that MCF7 cancer cells activate HIF-1α- and NFκB-driven luciferase reporters in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Consistent with these findings, acute knock-down of Cav-1 in stromal fibroblasts, using an siRNA approach, is indeed sufficient to induce autophagy, with the upregulation of both lysosomal and mitophagy markers. How does the loss of stromal Cav-1 and the induction of stromal autophagy affect cancer cell survival? Interestingly, we show that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptotic cell death. Thus, autophagic cancer-associated fibroblasts, in addition to providing recycled nutrients for cancer cell metabolism, also play a protective role in preventing the death of adjacent epithelial cancer cells. We demonstrate that cancer-associated fibroblasts upregulate the expression of TIGAR in adjacent epithelial cancer cells, thereby conferring resistance to apoptosis and autophagy. Finally, the mammary fat pads derived from Cav-1 (-/-) null mice show a hypoxia-like response in vivo, with the upregulation of autophagy markers, such as LC3 and BNIP3L. Taken together, our results provide direct support for the "Autophagic Tumor Stroma Model of Cancer Metabolism," and explain the exceptional prognostic value of a loss of stromal Cav-1 in cancer patients. Thus, a loss of stromal fibroblast Cav-1 is a biomarker for chronic hypoxia, oxidative stress and autophagy in the tumor microenvironment, consistent with its ability to predict early tumor recurrence, lymph node metastasis and tamoxifen-resistance in human breast cancers. Our results imply that cancer patients lacking stromal Cav-1 should benefit from HIF-inhibitors, NFκB-inhibitors, anti-oxidant therapies, as well as autophagy/lysosomal inhibitors. These complementary targeted therapies could be administered either individually or in combination, to prevent the onset of autophagy in the tumor stromal compartment, which results in a "lethal" tumor microenvironment.

HIF1-alpha functions as a tumor promoter in cancer-associated fibroblasts, and as a tumor suppressor in breast cancer cells

Our recent studies have mechanistically implicated a loss of stromal Cav-1 expression and HIF1-alpha-activation in driving the cancer-associated fibroblast phenotype, through the paracrine production of nutrients via autophagy and aerobic glycolysis. However, it remains unknown if HIF1a-activation is sufficient to confer the cancer-associated fibroblast phenotype. To test this hypothesis directly, we stably-expressed activated HIF1a in fibroblasts and then examined their ability to promote tumor growth using a xenograft model employing human breast cancer cells (MDA-MB-231). Fibroblasts harboring activated HIF1a showed a dramatic reduction in Cav-1 levels and a shift towards aerobic glycolysis, as evidenced by a loss of mitochondrial activity, and an increase in lactate production. Activated HIF1a also induced BNIP3 and BNIP3L expression, markers for the autophagic destruction of mitochondria. Most importantly, fibroblasts expressing activated HIF1a increased tumor mass by ~2-fold and tumor volume by ~3-fold, without a significant increase in tumor angiogenesis. In this context, HIF1a also induced an increase in the lymph node metastasis of cancer cells. Similar results were obtained by driving NFκB activation in fibroblasts, another inducer of autophagy. Thus, activated HIF1a is sufficient to functionally confer the cancer-associated fibroblast phenotype. It is also known that HIF1a expression is required for the induction of autophagy in cancer cells. As such, we next directly expressed activated HIF1a in MDA-MB-231 cells and assessed its effect on tumor growth via xenograft analysis. Surprisingly, activated HIF1a in cancer cells dramatically suppressed tumor growth, resulting in a 2-fold reduction in tumor mass and a 3-fold reduction in tumor volume. We conclude that HIF1a activation in different cell types can either promote or repress tumorigenesis. Based on these studies, we suggest that autophagy in cancer-associated fibroblasts promotes tumor growth via the paracrine production of recycled nutrients, which can directly "feed" cancer cells. Conversely, autophagy in cancer cells represses tumor growth via their "self-digestion." Thus, we should consider that the activities of various known oncogenes and tumor-suppressors may be compartment and cell-type specific, and are not necessarily an intrinsic property of the molecule itself. As such, other "classic" oncogenes and tumor suppressors will have to be re-evaluated to determine their compartment specific effects on tumor growth and metastasis. Lastly, our results provide direct experimental support for the recently proposed "Autophagic Tumor Stroma Model of Cancer."