Control of cancer-associated fibroblast function by oxidative stress: A new piece in the puzzle.

Fibroblasts are found in various proportions across the spectrum of carcinomas, constituting, in many cases, the preponderant cell population of the tumor stroma. Data from co-culture and reconstitution experiments indicate that fibroblasts have a prominent role in defining the rate and extent of cancer progression and metastasis. In a recent issue of Cell Cycle, Fiashi et al. described a new mechanism by which cancer-associated fibroblasts (CAFs) promote metastatic dissemination. They observed that CAFs have a major contribution to extracellular acidification through de novo carbonic anhydrase IX (CAIX) expression and activation upon stimulation by tumor epithelial cells. Importantly, such activation is observed in vitro as well as in CAFs isolated from patients bearing aggressive prostate carcinomas, confirming the physiopathological relevance of these observations. Thus, although the role of CAIX in epithelial tumor cells has been clearly established previously, those observations indicate that activation of CAIX in CAFs drives the extracellular acidification of prostate carcinoma microenvironment, and that CAIX represents a new marker for CAFs.

Since the hypoxia-inducible factor 1 (HIF-1) has been reported as a master CAIX regulator in tumor epithelial cells, the authors next investigated the effect of HIF-1 inhibition by pharmacological inhibitors or RNAi silencing. CAIX upregulation in stromal fibroblasts required a ROS-dependent stabilization of HIF-1 in normoxia. These findings are in line with previous reports describing redox-based HIF-1 stabilization under normoxic conditions, especially in CAFs associated with breast or prostate carcinomas.,

What are the functional consequences of CAIX-mediated extracellular acidification? CAIX activation resulted in increased MMP2 and MMP9 activity, leading to activation of EMT (as characterized by E-cadherin decrease, morphological features, and invasiveness) by tumor epithelial cells. Similarly, acidification of the extracellular medium greatly increased CAFs ability to drive EMT program in tumor cells. Importantly, CAIX-silenced CAFs were unable to support tumor outgrowth and lung metastasis formation upon co-injection with prostate tumor epithelial cells, confirming in vivo that CAIX is mandatory for the EMT process and metastatic dissemination.

This study is the first report of the role of CAFs in tumor microenvironment acidification, a salient feature commonly associated to tumor epithelial cells showing metabolic reprogramming toward the Warburg phenotype. Several biological processes are likely to contribute to such CAFs-mediated acidification. They include CAIX upregulation (described here) as well as increased glycolytic activity. Indeed, in contact with prostate tumor cells, CAFs undergo a mitochondrial oxidative stress and a metabolic reprogramming toward a Warburg phenotype, resulting in dramatic production of lactate that is extruded in the extracellular milieu together with H+ ions. CAIX upregulation in stromal cells is under the control of the HIF transcription factor, which accumulates through a ROS-dependent mechanism. The factors modulating ROS production in CAFs have not been investigated in Fiashi’s study and still remain unknown. However, one can argue that H2O2, a diffusible ROS involved in intercellular communication, might be produced by tumor epithelial cells and involved in CAIX activation in CAFs. In that matter, it has been reported that non-phagocytic NADPH-oxidase (Nox) enzymes are overexpressed at the plasma membrane of tumor epithelial cells and might contribute to the production of H2O2. Alternatively, MMPs, such as MMP3, have been shown to modulate activity of the mitochondrial respiratory chain, subsequently increasing cellular ROS content. It has also been shown that stimulation of receptor tyrosine kinase by growth factors, such as epidermal growth factor, is associated with ROS generation, suggesting that such growth factors might also contribute to redox-based HIF-1 stabilization. Lastly, when tumors reach a certain size, inadequate oxygen delivery leads to hypoxia. In response, new vessels are formed, allowing tissue reoxygenation. However, tumor blood vessels are mostly disorganized and leaky, and oxygen within tumors varies both in space and time. Cycles of hypoxia and re-oxygenation can increase ROS production, which can stabilize HIF-1 and further amplifies acidic stress.

Several CAIX inhibitors are currently in clinical development in solid tumors, including breast and kidney cancer. In this line, the current study supports efforts to treat cancer patients with CAIX inhibitors: as CAFs are non-transformed genetically stable cells and therefore less likely to acquire drug resistance, they represent ideal pharmacological targets. Further studies will be required to investigate combination therapies with conventional chemotherapeutic treatments.