Oncogenes induce the cancer-associated fibroblast phenotype: metabolic symbiosis and "fibroblast addiction" are new therapeutic targets for drug discovery

Abstract

Metabolic coupling, between mitochondria in cancer cells and catabolism in stromal fibroblasts, promotes tumor growth, recurrence, metastasis, and predicts anticancer drug resistance. Catabolic fibroblasts donate the necessary fuels (such as L-lactate, ketones, glutamine, other amino acids, and fatty acids) to anabolic cancer cells, to metabolize via their TCA cycle and oxidative phosphorylation (OXPHOS). This provides a simple mechanism by which metabolic energy and biomass are transferred from the host microenvironment to cancer cells. Recently, we showed that catabolic metabolism and "glycolytic reprogramming" in the tumor microenvironment are orchestrated by oncogene activation and inflammation, which originates in epithelial cancer cells. Oncogenes drive the onset of the cancer-associated fibroblast phenotype in adjacent normal fibroblasts via paracrine oxidative stress. This oncogene-induced transition to malignancy is "mirrored" by a loss of caveolin-1 (Cav-1) and an increase in MCT4 in adjacent stromal fibroblasts, functionally reflecting catabolic metabolism in the tumor microenvironment. Virtually identical findings were obtained using BRCA1-deficient breast and ovarian cancer cells. Thus, oncogene activation (RAS, NFkB, TGF-β) and/or tumor suppressor loss (BRCA1) have similar functional effects on adjacent stromal fibroblasts, initiating "metabolic symbiosis" and the cancer-associated fibroblast phenotype. New therapeutic strategies that metabolically uncouple oxidative cancer cells from their glycolytic stroma or modulate oxidative stress could be used to target this lethal subtype of cancers. Targeting "fibroblast addiction" in primary and metastatic tumor cells may expose a critical Achilles' heel, leading to disease regression in both sporadic and familial cancers.

Keywords: BRCA1; NFkB; RAS; TGF-beta; cancer-associated fibroblast; glycolysis; metabolic symbiosis; oncogene; oxidative stress; stromal biomarkers; tumor microenvironment; tumor suppressor.

Figure 1. Kaplan–Meier analysis of overall survival, using stromal Cav-1 and mct4 to predict clinical outcome. (A) TMA containing a cohort of 185 triple-negative breast cancer patients, with over 20 y of clinical follow-up data, was subjected to immunostaining with antibodies directed against Cav-1 and MCT4. Then, the expression levels of these two protein biomarkers were scored in the tumor stroma. Note that loss of Cav-1 and overexpression of MCT4 are strictly associated with poor clinical outcome. In contrast, patients with high stromal Cav-1 and absent MCT4 show >90% survival at >20 years post-diagnosis. Reproduced, with permission, from reference .

Figure 2. Epithelial oncogenes induce the cancer-associated fibroblast phenotype, in adjacent normal fibroblasts. Cav-1 and MCT4 as stromal biomarkers. (A) HaCaT cells (control [CTRL] vs. Ras-transformed [RAS]) were co-cultured with stromal fibroblasts. Then, the expression of Cav-1 and MCT4 was monitored by immunostaining. Note that Ras-transformed HaCaT cells specifically downregulate Cav-1 and upregulate MCT4 in adjacent fibroblasts, effectively functioning as reporters of the transition to malignancy in cancer cells. E, epithelia. (B) HaCaT cells (CTRL and RAS) were also cultured alone for comparison. Note that MCT4 is upregulated in Ras-transformed cells when they are cultured alone, but then downregulated when they are co-cultured with fibroblasts (as in A). Reproduced and modified, with permission, from reference .

Figure 3. Fibroblasts induce the expression of MCT1 in epithelial cancer cells to facilitate metabolic symbiosis. HaCaT–Ras cells were cultured alone (left) or co-cultured with fibroblasts (right) and then subjected to immunostaining with antibodies directed against MCT1. Note that co-culture with fibroblasts induces the expression of MCT1 in Ras-transformed epithelial cells. Thus, co-culture of Ras-transformed cells with fibroblasts induces reciprocal metabolic reprogramming, leading to metabolic symbiosis. Reproduced and modified, with permission, from reference .

Figure 4. Oncogenes drive oxidative stress and glycolysis in the tumor microenvironment. Summary illustrating that 2 divergent oncogenes (RAS and NFkB) use ROS production and cytokines (inflammation) to induce oxidative stress and glycolysis in adjacent cancer-associated fibroblasts (“the reverse Warburg effect”). Thus, oncogenes act at a distance, to metabolically reprogram the tumor microenvironment.

Figure 5. Diverse oncogenic stimuli induce the cancer-associated fibroblast phenotype via oxidative stress. Diagram illustrating that diverse oncogenic stimuli (RAS, NFkB, TGF-β, BRCA1 loss, ethanol exposure, ROS/hydrogen peroxide) all induce oxidative stress in the tumor microenvironment. This, in turn, promotes the catabolic cancer-associated fibroblast phenotype, resulting in a loss of Cav-1 and an increase in MCT4 expression. Treatment with N-acetyl-cysteine (NAC), a powerful antioxidant, is sufficient to reverse or prevent the cancer-associated fibroblast phenotype induced by these divergent oncogenic stressors.

Figure 6. BRCA1-deficient breast cancer cells induce the cancer-associated fibroblast phenotype. (A) BRCA1-deficient HCC cells were co-cultured with hTERT-immortalized fibroblasts. Then, expression of Cav-1 and MCT4 was monitored by immunostaining. Note that HCC cells drive a loss of stromal Cav-1 and the induction of stromal MCT4. Importantly, this CAF-phenotype was suppressed either by genetic replacement of the BRCA1 gene in the epithelial cancer cells, or by treatment with NAC, a powerful antioxidant. (B) Human breast cancer samples harboring BRCA1 mutations (9 out of 10 examined) also show a loss of stromal Cav-1 and the upregulation of MCT4, as well as strong mitochondrial staining with TOMM20, a marker of mitochondrial mass. For Cav-1 immunostaining, arrowheads point at blood vessels, which do not show a loss of Cav-1, in contrast to adjacent stromal fibroblasts. Images from one representative patient are shown. Reproduced and modified, with permission, from reference .

Figure 7. BRCA1-deficient ovarian cancer cells induce the cancer-associated fibroblast phenotype. (A) BRCA1-deficient UWB cells were co-cultured with hTERT-immortalized fibroblasts. Then, the expression of Cav-1 and MCT4 was monitored by immunostaining. Note that UWB cells drive a loss of stromal Cav-1 and the induction of stromal MCT4. (B) Importantly, this CAF-phenotype was suppressed by treatment with NAC, a powerful antioxidant. Genetic replacement of the BRCA1 gene in the epithelial cancer cells also suppressed the CAF-phenotype (not shown). Reproduced and modified, with permission, from reference .

Figure 8. BRCA1-deficient ovarian cancer cells induce an inflammatory phenotype in adjacent stromal fibroblasts. BRCA1-deficient UWB cells were co-cultured with NIH-3T3 fibroblasts, harboring an NFkB–luciferase reporter, to measure the activation of an inflammatory phenotype. Note that co-culture with BRCA1-deficient UWB cells activates NFkB-mediated gene transcription, in adjacent stromal fibroblasts. Furthermore, this pro-inflammatory phenotype was rescued by recombinant expression of the wild-type BRCA1 gene in UWB cells. The basal state of NFkB activation in NIH-3T3 fibroblasts cultured alone is shown for comparison. Day 0 actually represents 24 h of co-culture. Reproduced and modified, with permission, from reference .

Figure 9. Fibroblast addiction: Fibroblasts alleviate oncogenic stress in epithelial cancer cells. Catabolic fibroblasts provide high-energy fuels and precursors for biomass to adjacent cancer cells. As a consequence, fibroblasts protect cancer cells against stress by reducing ROS production, apoptosis, autophagy, and senescence in epithelial cancer cells. Experimental evidence indicates that this “fibroblast addiction” also confers drug resistance to anti-estrogens and other chemo-therapeutic agents by promoting oxidative mitochondrial metabolism in epithelial tumor cells.