Previous studies have demonstrated that a loss of Cav-1 in stromal cells induces the ligand-independent activation of the TGFβ pathway,7,40-42
with the increased transcription of the TGFβ target gene CTGF.1,4,14,43
It is now well known that CTGF induces tissue fibrosis, and that alterations in the extracellular matrix influence tumor growth and clinical outcome.21,44
It has also been demonstrated that a loss of stromal Cav-1 induces the metabolic reprogramming of cancer-associated fibroblasts with the induction of glycolysis and autophagy. However, it remains unknown if the activation of the TGFβ/CTGF pathway plays a role in the metabolic reprogramming of stromal cells induced by a loss of stromal Cav-1.
Therefore, the purpose of this study was to investigate if the TGFβ-target gene CTGF plays a role in the metabolic remodeling of the tumor microenvironment. In particular, we aimed to study if the cell type-specific expression of CTGF differentially affects tumor development.
The role of CTGF in breast cancer remains controversial. Elevated CTGF mRNA levels were found in human invasive ductal carcinomas and mouse mammary tumors and were confined to the fibrous tumor stroma.44
In another breast cancer study, overexpression of CTGF was positively associated with age, tumor size, stage and lymph node metastasis.45
Mechanistically, the tumor-promoting role of CTGF is supported by data showing that CTGF enhances tumor cell migration and angiogenesis46
and confers drug resistance.47,48
Conversely, other studies have indicated that CTGF may act as a tumor suppressor and have reported that low levels of CTGF are associated with increased metastasis and poor prognosis in breast cancer patients.49
For example, Hishikawa et al. demonstrated that forced overexpression of CTGF in MCF7 cells induces apoptosis.50
In our current studies, we propose a novel viewpoint to explain the controversial role of CTGF in breast cancer. Our data clearly indicate that CTGF exerts compartment-specific actions, and that its effects on tumor growth are opposite depending on the cell type producing CTGF. In fact, surprisingly, overexpression of CTGF in breast cancer epithelial cells inhibits tumor growth, but the opposite, tumor-promoting effect was observed when CTGF is overexpressed in the tumor fibroblast compartment.
We show for the first time that the overexpression of CTGF drives the induction of autophagy in both cell types, fibroblasts and breast cancer cells. Thus, CTGF-induced autophagy in fibroblasts can drive stromal cell digestion, leading to the release of chemical building blocks into the tumor microenvironment. These nutrients could be used as fuel for the anabolic growth of breast cancer cells, driving increased tumor mass independently of angiogenesis. Furthermore, we show that CTGF overexpression in stromal cells triggers the induction of glycolysis. The final product of glycolysis, L-lactate, could act in a paracrine way on breast cancer cells. Increased L-lactate uptake by breast cancer cells could activate LDH in cancer cells. At high lactate concentrations, LDH converts L-lactate into pyruvate, which is a substrate of the Krebs cycle, driving an increase in mitochondrial metabolic activity. Consistent with this hypothesis, we detected reductions in ATPase-IF1 expression in MDA-MB-231 cells co-cultured with CTGF fibroblasts compared with the control fibroblasts. Mechanistically, we show that the CTGF-mediated induction of autophagy occurs via increased oxidative stress and HIF-1α stabilization. Our results are consistent with previous studies showing that CTGF induces HIF-1α upregulation.51
However, the mechanism(s) by which CTGF induces HIF-1α activation is currently unknown.
Conversely, we show that forced CTGF overexpression in breast cancer cells inhibits tumor growth. We demonstrate that CTGF overexpression in epithelial breast cancer cells induces autophagy. Activation of autophagy in cancer cells increases tumor cell self-digestion, with a consequent decrease in tumor mass. Mechanistically, we propose that CTGF overexpression leads to increased oxidative stress, which, in turn, stabilizes HIF-1α. In fact, we have previously demonstrated that HIF-1α activation in breast cancer cells drives the induction of autophagy and inhibits tumor growth.8
Several studies have reported that increased intracellular ROS is involved in the induction of senescence. Two mechanisms have been proposed to explain ROS action on senescence. The first possibility is that ROS can lead to random damage to cellular components, thus acting as a non-specific senescence mediator. For example, an increase in ROS levels causes DNA damage, leading to activation of p53, which, in turn, drives cell cycle arrest via induction of p21. The second explanation is that ROS can function as messenger molecules that activate specific redox-dependent targets, and those could induce senescence.52
Recent evidence also links autophagy to cellular senescence. In particular, it has been demonstrated that ULK-3, the human homolog of the yeast ATG1 is essential for the initial building of the autophagosome, is highly expressed in senescent cells, and that ULK-3 overexpression induces autophagy and senescence. Furthermore, the knockdown of ATG5 or ATG7 reduces β-galactosidase activity, the most widely used marker of senescence.37
Inhibition of autophagy delays the senescence phenotype. Thus, the induction of autophagy in fibroblasts promotes the acquisition of the senescent phenotype.37
Recently, a new mechanism by which autophagy can lead to pre-mature senescence, has been proposed. Goligorsky et al. have demonstrated that stress-induced lysosomal membrane permeabilization drives the release of cathepsin B in the cytosol. Cathepsin B is a lysosomal cysteine protease, which induces SIRT1 depletion leading to autophagy-induced premature senescence.36
Thus, autophagy and senescence may be part of the same physiological process, known as the autophagy-senescence transition (AST) ().
Figure 11. CTGF drives the autophagy-senescence transition (ast) in cancer associated fibroblasts. Here, we show that activation of TGF-β signaling, via CTGF expression in normal fibroblasts, is sufficient to confer the cancer associated (more ...)
Cellular senescence is a reversible process that limits proliferation of cells at risk for neoplastic transformation and contributes to aging.53-56
On the other hand, although the mechanisms have not been fully elucidated yet but are likely to contrast aging, the induction of senescence leads to the secretion of several mitogenic substances, including growth factors, cytokines and extracellular matrix components,53,57
that alter the tumor microenvironment and favor tumor growth.58
In our study, we show for the first time that CTGF has the ability to induce senescence. Interestingly, CTGF-mediated induction of senescence is cell-type specific, as it occurs only in fibroblasts but not in breast cancer cells. It might be that the induction of senescence in fibroblasts could constitute an additional mechanism through which CTGF overexpression in stromal cells drives tumor growth. Although the molecular mechanism(s) that link autophagy with senescence are still unclear, we propose that systemic induction of autophagy and increased protein turnover could lead stromal cells to establish a senescent-like phenotype to protect them from further self-digestion.
Our results indicate that the tumor-promoting effects of CTGF may be independent of its well-known role in extracellular matrix remodeling. We unexpectedly observed that CTGF has opposite effects when it is produced by stromal cells or by breast cancer cells. This suggests that the CTGF effects are not due to its extracellular secretion; otherwise, we should observe the same results, independently of the cell type producing CTGF. Thus, our data clearly indicate that CTGF acts via an intracellular mechanism, likely through the metabolic reprogramming of the CTGF-producing cells. In support of this notion, we observed increased extracellular matrix deposition in tumor xenografts generated by CTGF-MDA-MB-231 cells and by CTGF-fibroblasts. Indeed, we observed increased extracellular matrix, which is usually considered a marker of tumor aggressiveness, in the CTGF-MDA-MB-231 xenogafts as well when the tumor mass was reduced. These data demonstrate that CTGF can still be secreted, but that the main CTGF tumor-promoting effects are due to its ability to drive metabolic reprogramming within cells. This is the first time that CTGF has been shown to modulate the metabolic status of stromal cells within the tumor microenvironment.
In conclusion, we propose a new compartment-specific role for CTGF in tumor formation, which is mediated via intracellular metabolic rearrangements. The overexpression of CTGF in breast cancer epithelial cells leads to autophagy activation, tumor cell digestion and inhibition of tumor growth. On the other hand, overexpression of CTGF in fibroblasts similarly drives the induction of autophagy, but in this case, enhances the release of recycled chemical building blocks into the tumor microenvironment, which can be used as “fuel” by anabolic tumor cells. Finally, the overexpression of CTGF drives a senescence phenotype in fibroblasts, which may further promote tumor growth.