We have previously shown using a co-culture model, that breast cancer cells induce oxidative stress in adjacent cancer associated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal Cav-1.39,40
However, the detailed signaling mechanism(s) underlying this process remain largely unknown. As loss of stromal Cav-1 is a powerful predictor of clinical outcome in human breast cancers,33–37
a clear understanding of the signaling mechanism(s) leading to the degradation of Cav-1 is of foremost importance.
Our data suggest a model whereby cancer cells trigger oxidative stress, and activate two pro-autophagic drivers, namely HIF-1α and NFκB, in adjacent stromal fibroblasts (). As a result, CAFs undergo autophagy and mitophagy, leading to loss of Cav-1 and metabolic re-programming. A loss of stromal Cav-1 aggravates oxidative stress and further promotes autophagy and mitophagy. Stromal autophagy generates building blocks (such as recycled free amino acids, fatty acids and nucleotides) that can be directly utilized by cancer cells to sustain growth and maintain cell viability. At the same time, HIF-1α activation and consequent mitophagy in CAFs induces mitochondrial dysfunction and enhances aerobic glycolysis, leading to the secretion of high-energy nutrients (such as lactate and pyruvate) that can directly feed oxidative mitochondrial metabolism in cancer cells. We also provide evidence that a loss of Cav-1 in stromal cells protects cancer cells from apoptosis, at least in part via TIGAR upregulation. Thus, our “working model” indicates that cancer cells exploit CAFs to satisfy their increased energy demand by forcing stromal cells to undergo a unilateral and vectorial energy transfer to sustain cancer cell growth. As such, catabolism in the tumor stroma promotes the anabolic growth of adjacent cancer cells, by providing a steady stream of recycled nutrients, independently of angiogenesis.
Figure 16 Autophagy in cancer associated fibroblasts (CAFs) fuels tumor cell survival. Here, we present a new model in which cancer cells trigger oxidative stress and activate two pro-autophagic drivers, namely HIF-1α and NFκB, in adjacent fibroblasts. (more ...)
We show here that three pro-autophagic stimuli are sufficient to induce a loss of Cav-1 in fibroblasts: (1) hypoxia, (2) hypoxia-mimetic drugs and (3) oxidative stress. To our knowledge, this is the first demonstration that hypoxia-induced autophagy leads to Cav-1 downregulation. A previous report had suggested that chronic myocardial hypoxia may decrease the expression of caveolin-3, the muscle-specific isoform of the caveolin gene family, as a way to increase nitric oxide production and protect against ischemia.47
Importantly, we show here that hypoxia-induced Cav-1 downregulation occurs by an autophagic mechanism and serves to protect adjacent cancer cells against apoptosis.
Concomitant with Cav-1 downregulation, we observe the upregulation of numerous autophagy and mitophagy markers. Treatment with the autophagic/lysosomal inhibitor chloroquine reverts the hypoxia-induced downregulation of Cav-1. We also show that activation of the autophagic molecular driver, HIF-1α, is sufficient to induce a loss of Cav-1 in fibroblasts. Interestingly, pharmacological activation of HIF-1α induces Cav-1 downregulation, whereas pharmacological inhibition of HIF-1α abrogates the hypoxia-induced degradation of Cav-1. Similarly, pharmacological inactivation of NFκB, another inducer of autophagy, prevents Cav-1 degradation. Moreover, treatment with an inducer of oxidative stress, namely BSO, is sufficient to induce the autophagic degradation of Cav-1. Thus, it appears that oxidative stress mediated via the induction of HIF1- and NFκB-activation drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we employed HIF-1α- and NFκB-driven luciferase reporters to monitor the ability of cancer cells to promote proautophagic signaling. Using this approach, we show that epithelial cancer cells promote the activation of HIF-1α and NFκB in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Thus, a loss of stromal Cav-1 in breast cancers is a biomarker of hypoxia, oxidative stress and autophagy in the tumor stromal microenvironment.
Little is known about HIF-1α activation in the tumor stromal compartment. It was recently shown that oxidative stress promotes myofibroblast differentiation through accumulation of HIF-1α and the chemokine CXCL12. Importantly, oxidative stress was shown to increase the migration of stromal fibroblasts, and to enhance tumor dissemination.48
We have also previously demonstrated that acute loss of Cav-1 in stromal fibroblasts is sufficient to drive HIF-1α accumulation, leading to mitochondrial dysfunction and aerobic glycolysis.39
Here, we show that HIF-1α activation in the stroma drives the autophagic degradation of Cav-1, providing a positive “feed-forward” control loop between HIF-1α activation and Cav-1 downregulation.
Activation of NFκB signaling in stromal macrophages is known to promote metastasis,49,50
however, the role of NFκB activity in CAFs is just starting to be unraveled. Previous studies have suggested that NFκB activation in CAFs may promote tumor growth. For example, stromal fibroblasts isolated from colorectal liver metastases display the upregulation of IL-8, a chemokine important for invasion and angiogenesis, via NFκB activation.51
In addition, transcriptome analysis has revealed that cancer-associated fibroblasts from breast, pancreas and skin cancers exhibit a pro-inflammatory gene signature compared to normal fibroblasts. Importantly, these inflammatory fibroblasts enhance tumor growth in an NFκB-dependent mechanism.52
We have previously shown that Cav-1 gene deletion in stromal cells drives the transcriptional activation of NFκB and of NFκB target genes.44
Importantly, we show here that cancer cells promote NF.B activation in adjacent fibroblasts. In addition, we provide evidence that pharmacological inactivation of NFκB prevents Cav-1 degradation in CAFs, and that acute loss of Cav-1 is sufficient to promote NFκB activation and autophagy, clearly indicating that in stromal cells, a loss of Cav-1 and NFκB activation can reciprocally promote each other.
Interestingly, our data indicate that acute loss of Cav-1 is sufficient to potently induce the expression of several autophagic and mitophagic markers. In addition, we show that in vivo, genetic ablation of Cav-1 in the mammary gland induces a pseudohypoxic state with increased autophagy. Our findings are consistent with recent studies suggesting that a loss of Cav-1 may promote autophagy. For example, transmission electron microscopy on murine myocardium has demonstrated increased assembly of autophagosomes in Cav-1 (−/−) null cardiac myocytes, as compared to WT controls.53
In addition, adipocytes from Cav-1 (−/−) null mice show increased LC3 staining and increased autophagic vacuoles.54
We show here that acute loss of Cav-1 is sufficient to trigger an autophagic response in fibroblasts. More importantly, our current data demonstrate that induction of autophagy via a Cav-1 deficiency may have important translational implications. We have recently proposed the “autophagic tumor stroma model of cancer metabolism,” whereby autophagy/mitophagy in the tumor stroma provides cancer cells with essential chemical building blocks (such as amino acids and nucleotides) to drive tumor progression and metastasis.46
Thus, a loss of stromal Cav-1 drives tumor progression by increasing “catabolism/autophagy” in the tumor microenvironment.
In direct support of our hypothesis, several studies have shown that overexpression of autophagic markers in the stroma, but not in tumor cells, correlates with poor clinical outcome in human cancers. For example, high stromal expression of ATG16L is associated with lympho-vascular invasion and lymph node metastasis in oral squamous cell carcinoma.55
In addition, stromal cathepsin K expression was found to be significantly higher in invasive breast cancers, compared with in situ carcinomas and in one patient set, it correlated with higher tumor stage.56
Similarly, in invasive breast ductal carcinoma, cathepsin D staining of tumor cells is associated with a lower nuclear grade and well-differentiated histology, whereas cathepsin D staining of stromal cells correlates with increased tumor size, poorly differentiated histology and shorter disease-free and overall survivals.57
Interestingly, cathepsin B was shown to be necessary for extracellular matrix degradation in v-Src transformed fibroblasts,58
and to drive proliferation and liver fibrosis in hepatic stellate cells.59
These data clearly suggest that the induction of an autophagic program in the tumor microenvironment correlates with poor clinical outcome and promotes aggressive cancer cell behavior.
What are the functional consequences of a loss of stromal Cav-1 and the induction of autophagy in the stromal compartment? Interestingly, we provide evidence that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptosis. Thus, autophagic cancer-associated fibroblasts, in addition to providing recycled nutrients to “feed” cancer cell metabolism, also play a protective role in preventing the death of adjacent epithelial cancer cells. Other studies have suggested that CAFs may promote tumor growth through decreased apoptosis. For example, conditioned media derived from CAFs expressing high levels of the GPI-anchored protein CD90 (Thy-1) was shown to protect prostate cancer cells from oxidative stress-induced apoptosis.60
Similarly, TGFβ1 treatment of prostate stromal cells was demonstrated to promote myofibroblast differentiation and to decrease apoptotic rates of co-cultured prostate cancer cells.61
We show here that loss of Cav-1 in stromal fibroblasts protects breast cancer cells from apoptosis. As Cav-1 is a potent inhibitor of TGFβ signaling, and we have previously shown that Cav-1 knockdown is sufficient to promote constitutive activation of TGFβ pathway,40,62
we speculate that TGFβ activation may play a key role in the loss of stromal Cav-1 and its enhancement of cancer cell survival.
Also, we provide evidence that increased expression of TIGAR may be one of the mechanism(s) by which cancer cells are protected against apoptosis. Co-culture with fibroblasts induces the compartment-specific expression of TIGAR in adjacent epithelial cancer cells. TIGAR is a newly discovered protein that protect cells against oxidative stress,43
and inhibits three related cellular processes, namely aerobic glycolysis, apoptosis and autophagy.42
Thus, TIGAR expression would render anabolic epithelial cancer cells resistant to the induction of aerobic glycolysis, apoptosis and autophagy, thereby facilitating mitochondrial biogenesis in cancer cells and promoting the transfer of nutrients from autophagic cancer-associated fibroblasts. These properties make TIGAR an ideal candidate to explain the “reverse Warburg effect,”63
and the “autophagic tumor stroma model of cancer metabolism.”46
In summary, we believe that during tumor formation, cancer cells and adjacent fibroblasts are metabolically coupled. Cancer cells induce a ROS-dependent loss of Cav-1 in adjacent fibroblasts via autophagic/lysosomal degradation. A loss of stromal Cav-1 then (1) promotes aerobic glycolysis and autophagy, thus producing nutrients (such as lactate and pyruvate) and chemical building blocks (such as amino acids, fatty acids and nucleotides) to “feed” adjacent cancer cells undergoing oxidative mitochondrial metabolism; and (2) protects cancer cells from apoptosis. Thus, upregulation of TIGAR in cancer cells could serve as a mechanism to promote cancer cell survival and prevent ROS generation, while inhibiting the two key metabolic processes, namely aerobic glycolysis and autophagy, in epithelial cancer cells that we show are activated in the tumor stroma. This model allows for the unilateral and vectorial transfer of nutrients from autophagy-prone catabolic stromal cells to autophagy-resistant anabolic cancer cells. Further studies will be required to fully address the role of stromal Cav-1 in modulating TIGAR expression in epithelial cancer cells.