Caveolin-1 and mitochondrial SOD2 (MnSOD) function as tumor suppressors in the stromal microenvironment: a new genetically tractable model for human cancer associated fibroblasts

Abstract

We have recently proposed a new model for understanding tumor metabolism, termed: "The Autophagic Tumor Stroma Model of Cancer Metabolism". In this new paradigm, catabolism (autophagy) in the tumor stroma fuels the anabolic growth of aggressive cancer cells. Mechanistically, tumor cells induce autophagy in adjacent cancer-associated fibroblasts via the loss of caveolin-1 (Cav-1), which is sufficient to promote oxidative stress in stromal fibroblasts. To further test this hypothesis, here we created human Cav-1 deficient immortalized fibroblasts using a targeted sh-RNA knock-down approach. Relative to control fibroblasts, Cav-1 deficient fibroblasts dramatically promoted tumor growth in xenograft assays employing an aggressive human breast cancer cell line, namely MDA-MB-231 cells. Co-injection of Cav-1 deficient fibroblasts, with MDA-MB-231 cells, increased both tumor mass and tumor volume by ~4-fold. Immuno-staining with CD31 indicated that this paracrine tumor promoting effect was clearly independent of angiogenesis. Mechanistically, proteomic analysis of these human Cav-1 deficient fibroblasts identified > 40 protein biomarkers that were upregulated, most of which were associated with i) myofibroblast differentiation, or ii) oxidative stress/hypoxia. In direct support of these findings, the tumor promoting effects of Cav-1 deficient fibroblasts could be functionally suppressed (nearly 2-fold) by the recombinant over-expression of SOD2 (superoxide dismutase 2), a known mitochondrial enzyme that de-activates superoxide, thereby reducing mitochondrial oxidative stress. In contrast, cytoplasmic soluble SOD1 had no effect, further highlighting a specific role for mitochondrial oxidative stress in this process. In summary, here we provide new evidence directly supporting a key role for a loss of stromal Cav-1 expression and oxidative stress in cancer-associated fibroblasts, in promoting tumor growth, which is consistent with "The Autophagic Tumor Stroma Model of Cancer". The human Cav-1 deficient fibroblasts that we have generated are a new genetically tractable model system for identifying other suppressors of the cancer-associated fibroblast phenotype, via a genetic "complementation" approach. This has important implications for understanding the pathogenesis of triple negative and basal breasts cancers, as well as tamoxifen-resistance in ER+ breast cancers, which are all associated with a Cav-1 deficient "lethal" tumor micro-environment, driving poor clinical outcome.

Figure 1

Targeted knock-down of Cav-1 protein expression in hTERT-fibroblasts. To dissect the role of Cav-1 in promoting the growth of triple negative breast cancers, we have created a matched set of hTERT-immortalized human fibroblast cell lines (from parental hTERT-BJ1), either expressing an shRNA targeting Cav-1 or a control shRNA. This retroviral vector also contains GFP, so transduced cells were recovered by FACS sorting. Successful knock-down of Cav-1 was verified by western blot analysis. The expression of beta-actin is shown as a control for equal protein loading. Ctl, control sh-RNA; sh-Cav1, harboring sh-RNA targeting Cav-1.

Figure 2

Targeted knock-down of Cav-1 in stromal fibroblasts dramatically promotes breast cancer tumor growth. Control or Cav-1 knock-down fibroblasts (300,000 cells) were co-injected with MDA-MB-321 cells (1 million cells) in the flanks of nude mice. After 4.5 weeks post-injection, the tumors were harvested. Note that relative to control fibroblasts, Cav-1 knock-down fibroblasts increased tumor mass by ∼4-fold (A) and increased tumor volume by ∼4-fold (B). An asterisk indicates that p ≤ 0.01. Fibroblasts injected alone did not form tumors. MDA-MB-231 cells injected alone, behaved as MDA-231 cells injected with control fibroblasts. N = 20 flank injections for each experimental group. Ctl, control sh-RNA; sh-Cav1, harboring sh-RNA targeting Cav-1.

Figure 3

Targeted knock-down of Cav-1 in stromal fibroblasts does not affect tumor angiogenesis. Frozen sections from the tumors were cut and immuno-stained with anti-CD31 antibodies, and vessel density was quantitated (A). Note that no significant increases in vessel density were observed, suggesting that the tumor promoting effects of the Cav-1 knock-down fibroblasts we observe are independent of angiogenesis (n.s., not significant). Representative images are shown in (B). Ctl, control sh-RNA; KD, harboring sh-RNA targeting Cav-1 (knock-down).

Figure 4

Recombinant overexpression of eNOS in fibroblasts does not promote tumor growth. Control or eNOS-overexpressing fibroblasts (300,000 cells) were co-injected with MDA-MB-321 cells (1 million cells) in the flanks of nude mice. After 4 weeks post-injection, the tumors were harvested. Note that no significant differences were noted between control fibroblasts and eNOS-overexpressing fibroblasts. N = 10 flank injections for each experimental group. n.s., not significant. EV, empty vector; eNOS, stably overexpressing eNOS. (A) tumor weight; (B) tumor volume.

Figure 5

Mitochondrial SOD2 significantly reverts the tumor promoting phenotype of Cav-1 deficient fibroblasts. We have previously shown that loss of Cav-1 increases ROS production in stromal fibroblasts. To combat the resulting oxidative stress, we stably overexpressed SOD2 in Cav-1 knock-down fibroblasts, using a lenti-viral vector with puromycin resistance. Cav-1 knock-down cells were transfected with the empty vector alone, in parallel. Then, these two fibroblast lines were co-injected with MDA-MB-231 cells into the flanks of nude mice. Note that overexpression of SOD2, a mitochondrial enzyme that deactivates super-oxide, is sufficient to reduce the tumor promoting effects of Cav-1 knock-down fibroblasts by nearly 2-fold. An asterisk indicates that p = 0.01 (B). The overexpression of SOD2 was validated by western blot analysis (A). The expression of b-actin is shown as a control for equal protein loading. N ≥ 9 flank injections for each experimental group.

Figure 6

Cytoplasmic soluble SOD1 does not revert the tumor promoting phenotype of Cav-1 deficient fibroblasts. To combat the oxidative stress, we stably overexpressed SOD1 in Cav-1 knock-down fibroblasts, using a lenti-viral vector with puromycin resistance. Cav-1 knock-down cells were transfected with the empty vector alone, in parallel. Then, these two fibroblast lines were co-injected with MDA-MB-231 cells into the flanks of nude mice. Note that overexpression of SOD1, a cytoplasmic soluble enzyme that deactivates super-oxide, is not sufficient to reduce the tumor promoting effects of Cav-1 knock-down fibroblasts. The overexpression of SOD1 was validated by western blot analysis (A). The expression of β-actin is shown as a control for equal protein loading. N ≥ 8 flank injections for each experimental group (n.s., not significant).