Caveolin-1 Expression Level in Cancer Associated Fibroblasts Predicts Outcome in Gastric Cancer

Aims

Altered expression of epithelial or stromal caveolin-1 (Cav-1) is observed in various types of human cancers. However, the clinical significance of Cav-1 expression in gastric cancer (GC) remains largely unknown. The present study aims to explore the clinicopathological significance and prognostic value of both tumor cells and cancer associated fibroblasts (CAFs) Cav-1 in GC.

Methods and Results

Quantum dots immunofluorescence histochemistry was performed to examine the expression of Cav-1 in 20 cases of gastritis without intestinal metaplasia (IM), 20 cases of gastritis with IM and 286 cases of GC. Positive rates of epithelial Cav-1 in gastritis without IM, gastritis with IM and GC showed a decreasing trend (P = 0.012). Low expression of Cav-1 in CAFs but not in tumor cells was an independent predictor of poor prognosis in GC patients (P = 0.034 and 0.005 respectively in disease free survival and overall survival). Cav-1 level in tumor cells and CAFs showed no significant correlation with classic clinicopathological features.

Conclusions

Loss of epithelial Cav-1 may promote malignant progression and low CAFs Cav-1 level herald worse outcome of GC patient, suggesting CAFs Cav-1 may be a candidate therapeutic target and a useful prognostic marker of GC.     

LncRNA MALAT1 promotes gastric cancer progression via inhibiting autophagic flux and inducing fibroblast activation

Autophagy defection contributes to inflammation dysregulation, which plays an important role in gastric cancer (GC) progression. Various studies have demonstrated that long noncoding RNA could function as novel regulators of autophagy. Previously, long noncoding RNA MALAT1 was reported upregulated in GC cells and could positively regulate autophagy in various cancers. Here, we for the first time found that MALAT1 could promote interleukin-6 (IL-6) secretion in GC cells by blocking autophagic flux. Moreover, IL-6 induced by MALAT1 could activate normal to cancer-associated fibroblast conversion. The interaction between GC cells and cancer-associated fibroblasts in the tumour microenvironment could facilitate cancer progression. Mechanistically, MALAT1 overexpression destabilized the PTEN mRNA in GC cells by competitively interacting with the RNA-binding protein ELAVL1 to activate the AKT/mTOR pathway for impairing autophagic flux. As a consequence of autophagy inhibition, SQSTM1 accumulation promotes NF-κB translocation to elevate IL-6 expression. Overall, these results demonstrated that intercellular interaction between GC cells and fibroblasts was mediated by autophagy inhibition caused by increased MALAT1 that promotes GC progression, providing novel prevention and therapeutic strategies for GC.Subject terms: Gastric cancer, Long non-coding RNAs     

CPNE8 Promotes Gastric Cancer Metastasis by Modulating Focal Adhesion Pathway and Tumor Microenvironment

Little is known about the oncogenic role or biological function of copine Ⅷ (CPNE8) in gastric cancer (GC). Based on TCGA database, we screened for CPNE8 and analyzed the expression of CPNE8 in GC. The correlations between CPNE8 and clinical features were analyzed using TCGA and GEO databases. The prognostic value of CPNE8 was assessed using Cox analysis and Kaplan-Meier curves. The results showed that increased expression of CPNE8 was positively correlated with metastasis and can be considered an independent prognostic risk factor for poor survival. We found that CPNE8 can promote cell proliferation, migration, and invasiveness in GC using in vitro and in vivo experiments. Our study demonstrated that CPNE8 promotes tumor progression via regulation of focal adhesion, and these effects can be rescued by focal adhesion kinase (FAK) inhibitor GSK2256098 or knockdown of FAK. In addition, CPNE8 was correlated significantly with the infiltration of cancer-associated fibroblasts and immune cells, as demonstrated by various algorithms, and high CPNE8 expression predicted poor efficacy of immune checkpoint therapy. Our findings suggest that CPNE8 modulates focal adhesion and tumor microenvironment to promote GC progression and invasiveness and could serve as a novel prognostic biomarker in GC.     

Epigenetic silencing of microRNA-149 in cancer-associated fibroblasts mediates prostaglandin E2/interleukin-6 signaling in the tumor microenvironment

Tumor initiation and growth depend on its microenvironment in which cancer-associated fibroblasts (CAFs) in tumor stroma play an important role. Prostaglandin E2 (PGE2) and interleukin (IL)-6 signal pathways are involved in the crosstalk between tumor and stromal cells. However, how PGE2-mediated signaling modulates this crosstalk remains unclear. Here, we show that microRNA (miR)-149 links PGE2 and IL-6 signaling in mediating the crosstalk between tumor cells and CAFs in gastric cancer (GC). miR-149 inhibited fibroblast activation by targeting IL-6 and miR-149 expression was substantially suppressed in the CAFs of GC. miR-149 negatively regulated CAFs and their effect on GC development both in vitro and in vivo. CAFs enhanced epithelial-to-mesenchymal transition (EMT) and the stem-like properties of GC cells in a miR-149-IL-6-dependent manner. In addition to IL-6, PGE2 receptor 2 (PTGER2/EP2) was revealed as another potential target of miR-149 in fibroblasts. Furthermore, H. pylori infection, a leading cause of human GC, was able to induce cyclooxygenase-2 (COX-2)/PGE2 signaling and to enhance PGE2 production, resulting in the hypermethylation of miR-149 in CAFs and increased IL-6 secretion. Our findings indicate that miR-149 mediates the crosstalk between tumor cells and CAFs in GC and highlight the potential of interfering miRNAs in stromal cells to improve cancer therapy.     

Autophagy in cancer associated fibroblasts promotes tumor cell survival: Role of hypoxia,HIF1 induction and NFκB activation in the tumor stromal microenvironment

Recently, using a co-culture system, we demonstrated that MCF7 epithelial cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal caveolin-1 (Cav-1). However, the detailed signaling mechanism(s) underlying this process remain largely unknown. Here, we show that hypoxia is sufficient to induce the autophagic degradation of Cav-1 in stromal fibroblasts, which is blocked by the lysosomal inhibitor chloroquine. Concomitant with the hypoxia-induced degradation of Cav-1, we see the upregulation of a number of well-established autophagy/mitophagy markers, namely LC3, ATG16L, BNIP3, BNIP3L, HIF-1α and NFκB. In addition, pharmacological activation of HIF-1α drives Cav-1 degradation, while pharmacological inactivation of HIF-1 prevents the downregulation of Cav-1. Similarly, pharmacological inactivation of NFκB—another inducer of autophagy—prevents Cav-1 degradation. Moreover, treatment with an inhibitor of glutathione synthase, namely BSO, which induces oxidative stress via depletion of the reduced glutathione pool, is sufficient to induce the autophagic degradation of Cav-1. Thus, it appears that oxidative stress mediated induction of HIF1- and NFκB-activation in fibroblasts drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we show that MCF7 cancer cells activate HIF-1α- and NFκB-driven luciferase reporters in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Consistent with these findings, acute knockdown of Cav-1 in stromal fibroblasts, using an siRNA approach, is indeed sufficient to induce autophagy, with the upregulation of both lysosomal and mitophagy markers. How does the loss of stromal Cav-1 and the induction of stromal autophagy affect cancer cell survival? Interestingly, we show that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptotic cell death. Thus, autophagic cancer-associated fibroblasts, in addition to providing recycled nutrients for cancer cell metabolism, also play a protective role in preventing the death of adjacent epithelial cancer cells. We demonstrate that cancer-associated fibroblasts upregulate the expression of TIGAR in adjacent epithelial cancer cells, thereby conferring resistance to apoptosis and autophagy. Finally, the mammary fat pads derived from Cav-1 (−/−) null mice show a hypoxia-like response in vivo, with the upregulation of autophagy markers, such as LC3 and BNIP3L. Taken together, our results provide direct support for the “autophagic tumor stroma model of cancer metabolism”, and explain the exceptional prognostic value of a loss of stromal Cav-1 in cancer patients. Thus, a loss of stromal fibroblast Cav-1 is a biomarker for chronic hypoxia, oxidative stress and autophagy in the tumor microenvironment, consistent with its ability to predict early tumor recurrence, lymph node metastasis and tamoxifen-resistance in human breast cancers. Our results imply that cancer patients lacking stromal Cav-1 should benefit from HIF-inhibitors, NFκB-inhibitors, anti-oxidant therapies, as well as autophagy/lysosomal inhibitors. These complementary targeted therapies could be administered either individually or in combination, to prevent the onset of autophagy in the tumor stromal compartment, which results in a “lethal” tumor microenvironment.Key words: caveolin-1, autophagy, BNIP3, cancer-associated fibroblasts, HIF1, hypoxia, LC3, mitophagy, NFκB, oxidative stress, predictive biomarker, TIGAR, tumor stroma     

Hypomethylation of repetitive elements in blood leukocyte DNA and risk of gastric lesions in a Chinese population

BackgroundTo explore the association between hypomethylation of repetitive elements (LINE-1, Sat2, and ALU) in blood leukocyte DNA and risks of gastric lesions, and development of gastric cancer (GC), a population-based study was conducted in a high-risk area of GC in China.MaterialsMethylation levels were determined by MethyLight in 902 subjects with various gastric lesions from two cohort studies at baseline and 276 subjects with long-term follow-up data.ResultsThe frequency of LINE-1 or Sat2 hypomethylation was significantly increased in subjects with dysplasia (DYS) compared with superficial gastritis/chronic atrophic gastritis. The odds ratios (ORs) were 2.22 [95% confidence interval (CI): 1.45–3.40] for LINE-1 and 1.58 (95% CI: 1.14–2.21) for Sat2. A dose–response pattern was found for the risk of DYS and LINE-1 hypomethylation (P-trend < 0.001). Further stratified analysis indicated that the frequency of LINE-1 or Sat2 hypomethylation was higher in subjects with Helicobacter pylori infection. The ORs were 1.83 (95% CI: 1.12–2.99) for LINE-1 and 1.44 (95% CI: 1.01–2.05) for Sat2. The follow-up data indicated that the risk of progression to GC was increased in intestinal metaplasia (IM) subjects with LINE-1 hypomethylation (OR = 2.82; 95% CI: 1.17–6.77) or Sat2 hypomethylation (OR = 2.78; 95% CI: 1.15–6.74). The risk of progression to GC was also increased in DYS subjects with Sat2 hypomethylation (OR = 5.24; 95% CI: 2.00–13.74).ConclusionsThese findings suggest that hypomethylation of repetitive elements in blood leukocytes is associated with the risks of advanced gastric lesions and development of GC.     

Identification of CEACAM5 as a Biomarker for Prewarning and Prognosis in Gastric Cancer

MGd1, a monoclonal antibody raised against gastric cancer cells, possesses a high degree of specificity for gastric cancer (GC). Here we identified that the antigen of MGd1 is CEACAM5, and used MGd1 to investigate the expression of CEACAM5 in non-GC and GC tissues (N=643), as a biomarker for prewarning and prognosis. The expression of CEACAM5 was detected by immunohistochemistry in numerous tissues; its clinicopathological correlation was statistically analyzed. CEACAM5 expression was increased progressively from normal gastric mucosa to chronic atrophic gastritis, intestinal metaplasia, dysplasia and finally to GC (p<0.05). In gastric precancerous lesions (intestinal metaplasia and dysplasia), CEACAM5-positive patients had a higher risk of developing GC as compared with CEACAM5-negative patients (OR = 12.68, p<0.001). Besides, CEACAM5 was found positively correlated with invasion depth of gastric adenocarcinoma (p<0.001). In survival analysis, CEACAM5 was demonstrated to be an independent prognostic predictor for patients with GC of clinical stage IIIA/IV (p=0.033). Our results demonstrate that CEACAM5 is a promising biomarker for GC prewarning and prognostic evaluation.     

Autophagy in cancer associated fibroblasts promotes tumor cell survival

Recently, using a co-culture system, we demonstrated that MCF7 epithelial cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal caveolin-1 (Cav-1). However, the detailed signaling mechanism(s) underlying this process remain largely unknown. Here, we show that hypoxia is sufficient to induce the autophagic degradation of Cav-1 in stromal fibroblasts, which is blocked by the lysosomal inhibitor chloroquine. Concomitant with the hypoxia-induced degradation of Cav-1, we see the upregulation of a number of well-established autophagy/mitophagy markers, namely LC3, ATG16L, BNIP3, BNIP3L, HIF-1α and NFκB. In addition, pharmacological activation of HIF-1α drives Cav-1 degradation, while pharmacological inactivation of HIF-1 prevents the downregulation of Cav-1. Similarly, pharmacological inactivation of NFκB-another inducer of autophagy-prevents Cav-1 degradation. Moreover, treatment with an inhibitor of glutathione synthase, namely BSO, which induces oxidative stress via depletion of the reduced glutathione pool, is sufficient to induce the autophagic degradation of Cav-1. Thus, it appears that oxidative stress mediated induction of HIF1- and NFκB-activation in fibroblasts drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we show that MCF7 cancer cells activate HIF-1α- and NFκB-driven luciferase reporters in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Consistent with these findings, acute knock-down of Cav-1 in stromal fibroblasts, using an siRNA approach, is indeed sufficient to induce autophagy, with the upregulation of both lysosomal and mitophagy markers. How does the loss of stromal Cav-1 and the induction of stromal autophagy affect cancer cell survival? Interestingly, we show that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptotic cell death. Thus, autophagic cancer-associated fibroblasts, in addition to providing recycled nutrients for cancer cell metabolism, also play a protective role in preventing the death of adjacent epithelial cancer cells. We demonstrate that cancer-associated fibroblasts upregulate the expression of TIGAR in adjacent epithelial cancer cells, thereby conferring resistance to apoptosis and autophagy. Finally, the mammary fat pads derived from Cav-1 (-/-) null mice show a hypoxia-like response in vivo, with the upregulation of autophagy markers, such as LC3 and BNIP3L. Taken together, our results provide direct support for the "Autophagic Tumor Stroma Model of Cancer Metabolism," and explain the exceptional prognostic value of a loss of stromal Cav-1 in cancer patients. Thus, a loss of stromal fibroblast Cav-1 is a biomarker for chronic hypoxia, oxidative stress and autophagy in the tumor microenvironment, consistent with its ability to predict early tumor recurrence, lymph node metastasis and tamoxifen-resistance in human breast cancers. Our results imply that cancer patients lacking stromal Cav-1 should benefit from HIF-inhibitors, NFκB-inhibitors, anti-oxidant therapies, as well as autophagy/lysosomal inhibitors. These complementary targeted therapies could be administered either individually or in combination, to prevent the onset of autophagy in the tumor stromal compartment, which results in a "lethal" tumor microenvironment.     

Phosphocaveolin-1 Enforces Tumor Growth and Chemoresistance in Rhabdomyosarcoma

Caveolin-1 (Cav-1) can ambiguously behave as either tumor suppressor or oncogene depending on its phosphorylation state and the type of cancer. In this study we show that Cav-1 was phosphorylated on tyrosine 14 (pCav-1) by Src-kinase family members in various human cell lines and primary mouse cultures of rhabdomyosarcoma (RMS), the most frequent soft-tissue sarcoma affecting childhood. Cav-1 overexpression in the human embryonal RD or alveolar RH30 cells yielded increased pCav-1 levels and reinforced the phosphorylation state of either ERK or AKT kinase, respectively, in turn enhancing in vitro cell proliferation, migration, invasiveness and chemoresistance. In contrast, reducing the pCav-1 levels by administration of a Src-kinase inhibitor or through targeted Cav-1 silencing counteracted the malignant in vitro phenotype of RMS cells. Consistent with these results, xenotransplantation of Cav-1 overexpressing RD cells into nude mice resulted in substantial tumor growth in comparison to control cells. Taken together, these data point to pCav-1 as an important and therapeutically valuable target for overcoming the progression and multidrug resistance of RMS.     

Oxidative stress in cancer associated fibroblasts drives tumor-stroma co-evolution: A new paradigm for understanding tumor metabolism,the field effect and genomic instability in cancer cells

Loss of stromal fibroblast caveolin-1 (Cav-1) is a powerful single independent predictor of poor prognosis in human breast cancer patients, and is associated with early tumor recurrence, lymph node metastasis and tamoxifen-resistance. We developed a novel co-culture system to understand the mechanism(s) by which a loss of stromal fibroblast Cav-1 induces a “lethal tumor microenvironment.” Here, we propose a new paradigm to explain the powerful prognostic value of stromal Cav-1. In this model, cancer cells induce oxidative stress in cancer-associated fibroblasts, which then acts as a “metabolic” and “mutagenic” motor to drive tumor-stroma co-evolution, DNA damage and aneuploidy in cancer cells. More specifically, we show that an acute loss of Cav-1 expression leads to mitochondrial dysfunction, oxidative stress and aerobic glycolysis in cancer associated fibroblasts. Also, we propose that defective mitochondria are removed from cancer-associated fibroblasts by autophagy/mitophagy that is induced by oxidative stress. As a consequence, cancer associated fibroblasts provide nutrients (such as lactate) to stimulate mitochondrial biogenesis and oxidative metabolism in adjacent cancer cells (the “Reverse Warburg effect”). We provide evidence that oxidative stress in cancer-associated fibroblasts is sufficient to induce genomic instability in adjacent cancer cells, via a bystander effect, potentially increasing their aggressive behavior. Finally, we directly demonstrate that nitric oxide (NO) over-production, secondary to Cav-1 loss, is the root cause for mitochondrial dysfunction in cancer associated fibroblasts. In support of this notion, treatment with anti-oxidants (such as N-acetyl-cysteine, metformin and quercetin) or NO inhibitors (L-NAME) was sufficient to reverse many of the cancer-associated fibroblast phenotypes that we describe. Thus, cancer cells use “oxidative stress” in adjacent fibroblasts (1) as an “engine” to fuel their own survival via the stromal production of nutrients and (ii) to drive their own mutagenic evolution towards a more aggressive phenotype, by promoting genomic instability. We also present evidence that the “field effect” in cancer biology could also be related to the stromal production of ROS and NO species. eNOS-expressing fibroblasts have the ability to downregulate Cav-1 and induce mitochondrial dysfunction in adjacent fibroblasts that do not express eNOS. As such, the effects of stromal oxidative stress can be laterally propagated, amplified and are effectively “contagious”—spread from cell-to-cell like a virus—creating an “oncogenic/mutagenic” field promoting widespread DNA damage.Key words: caveolin-1, cancer associated fibroblasts, oxidative stress, reactive oxygen species (ROS), mitochondrial dysfunction, autophagy, nitric oxide (NO), DNA damage, aneuploidy, genomic instability, anti-oxidant cancer therapy, the “field effect” in cancer biology     

Caveolin-1 inhibits oligodendroglial differentiation of neural stem/progenitor cells through modulating β-catenin expression

In the present study, we aim to elucidate the role of caveolin-1 (Cav-1) in modulating oligodendroglial differentiation of neural progenitor cells (NPCs) in vivo and in vitro. For in vivo experiments, we investigated oligodendroglial differentiation by detecting the expressions of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) and β-catenin in the brains of wild type mice and Cav-1 knockout mice. Cav-1 knockout mice revealed more oligodendroglial differentiation, but lower levels of β-catenin expression than wild type mice. For in vitro experiments, we observed the potential roles of Cav-1 in modulating β-catenin expression and oligodendroglial differentiation in isolated cultured NPCs by manipulating Cav-1 expression with Cav-1 scaffolding domain peptide and Cav-1 RNA silencing approach. In the differentiating NPCs, Cav-1 scaffolding domain peptide markedly inhibited oligodendroglial formation, but up-regulated the expression of β-catenin. In contrast, the knockdown of Cav-1 promoted oligodendroglial differentiation of NPCs, but down-regulated the expression of β-catenin. Taken together, these results directly prove that caveolin-1 can inhibit oligodendroglial differentiation of NPCs through modulating β-catenin expression.     

Serum OPN Expression for Identification of Gastric Cancer and Atrophic Gastritis and Its Influencing Factors

Background

Most studies have found that osteopontin (OPN) expression level is related to the poor prognosis of gastric cancer. However, few studies have examined the relationship between OPN expression and gastric precancerous diseases, and the potential role of OPN in the formation and development of GC. We investigated the relationships between serum OPN levels and the risks of gastric cancer (GC) and its precancerous disease, to explore the diagnostic efficacy of serum OPN level for GC and atrophic gastritis and its influencing factors.

Methods

A total of 1,452 patients were enrolled, including 609 with mild superficial gastritis (SG), 594 with atrophic gastritis (AG) and 249 with GC. The levels of serum OPN and serum Helicobacter pylori IgG antibody were detected by enzyme-linked immunosorbent assay.

Results

Serum OPN levels increased from mild SG (1.99±1.91 ng/ml) to AG (2.37±2.27 ng/ml) to GC (5.94±4.52 ng/ml) (P≤0.002), along with increasing severity of gastric disease. OPN levels were significantly higher in patients with GC compared with the non-cancer population (2.17±2.10, P<0.0001). Serum OPN level was positively correlated with age and was higher in men than women, but was not correlated with H. pylori infection status. The area under the receiver operating characteristic curve was 0.805, the optimal cutoff was 2.56 ng/ml and the sensitivity and specificity were 74.3% and 71.8%, respectively, for the ability of serum OPN to discriminate GC.

Conclusions

Serum OPN expression was closely related to the risks of GC and AG, and it might be a useful marker for the discrimination of GC. OPN level was positively correlated with age and male sex, but was not affected by H. pylori infection, and it was promoted by smoking and drinking, in patients with mild SG.     

A New Polymorphism Biomarker rs629367 Associated with Increased Risk and Poor Survival of Gastric Cancer in Chinese by Up-Regulated miRNA-let-7a Expression

Background

Variant in pri-miRNA could affect miRNA expression and mature process or splicing efficiency, thus altering the hereditary susceptibility and prognosis of cancer. We aimed to assess miRNA-let-7 single nucleotide polymorphisms (SNP) associated with the risk and prognosis of gastric cancer (GC) as predicting biomarkers, and furthermore, its possible mechanisms.

Methods

A two-stage case-control study was designed to screen four miRNA SNPs (pri-let-7a-2 rs629367 and rs1143770, pri-let-7a-1 rs10739971, pri-let-7f-2 rs17276588) in 107 GC patients, 107 atrophic gastritis (AG), and matched 124 controls using PCR-RFLP. Two promising SNPs were validated in another independent 1949 samples (including 579 gastric cancer patients, 649 atrophic gastritis and 721 controls) using Sequenom MassARRAY platform and sequencing.

Results

We found that pri-let-7a-2 rs629367 CC variant genotype was associated with increased risks of gastric cancer and atrophic gastritis by 1.83-fold and 1.86-fold, respectively. For gastric cancer prognosis, patients with rs629367 CC genotype had significantly poorer survival than patients with AA genotype (log-rank P = 0.004). We further investigated the let-7a expression levels in serum and found that let-7a expression elevated gradually for rs629367 AA, CA, CC genotype in the atrophic gastritis group (P = 0.043). Furthermore, we confirmed these findings in vitro study by overexpressing let-7a carrying pri-let-7a-2 wild-type A or polymorphic-type C allele (P<0.001).

Conclusions

pri-let-7a-2 rs629367 CC genotype could increase the risks of gastric cancer as well as atrophic gastritis and was also associated with poor survival of gastric cancer, which possibly by affecting the mature let-7a expression, and could serve as a predicting biomarker for high-risk and poor prognosis of gastric cancer.     

Genetic ablation of caveolin-2 sensitizes mice to bleomycin-induced injury

Caveolar domains act as platforms for the organization of molecular complexes involved in signal transduction. Caveolin proteins, the principal structural components of caveolae, have been involved in many cellular processes. Caveolin-1 (Cav-1) and caveolin-2 (Cav-2) are highly expressed in the lung. Cav-1-deficient mice (Cav-1^−/−) and Cav-2-deficient mice (Cav-2^−/−) exhibit severe lung dysfunction attributed to a lack of Cav-2 expression. Recently, Cav-1 has been shown to regulate lung fibrosis in different models. Here, we show that Cav-2 is also involved in modulation of the fibrotic response, but through distinct mechanisms. Treatment of wild-type mice with the pulmonary fibrosis-inducer bleomycin reduced the expression of Cav-2 and its phosphorylation at tyrosine 19. Importantly, Cav-2^−/− mice, but not Cav-1^−/− mice, were more sensitive to bleomycin-induced lung injury in comparison to wild-type mice. Bleomycin-induced lung injury was characterized by alveolar thickening, increase in cell density, and extracellular matrix deposition. The lung injury observed in bleomycin-treated Cav-2^−/− mice was not associated with alterations in the TGF-β signaling pathway and/or in the ability to produce collagen. However, apoptosis and proliferation were more prominent in lungs of bleomycin-treated Cav-2^−/− mice. Since Cav-1^−/− mice also lack Cav-2 expression and show a different outcome after bleomycin treatment, we conclude that Cav-1 and Cav-2 have distinct roles in bleomycin induced-lung fibrosis, and that the balance of both proteins determines the development of the fibrotic process.     

Matrix remodeling stimulates stromal autophagy, “fueling” cancer cell mitochondrial metabolism and metastasis

We have previously demonstrated that loss of stromal caveolin-1 (Cav-1) in cancer-associated fibroblasts is a strong and independent predictor of poor clinical outcome in human breast cancer patients. However, the signaling mechanism(s) by which Cav-1 downregulation leads to this tumor-promoting microenvironment are not well understood. To address this issue, we performed an unbiased comparative proteomic analysis of wild-type (WT) and Cav-1^-/- null mammary stromal fibroblasts (MSFs). Our results show that plasminogen activator inhibitor type 1 and type 2 (PAI-1 and PAI-2) expression is significantly increased in Cav-1^-/- MSFs. To establish a direct cause-effect relationship, we next generated immortalized human fibroblast lines stably overexpressing either PAI-1 or PAI-2. Importantly, PAI-1/2(+) fibroblasts promote the growth of MDA-MB-231 tumors (a human breast cancer cell line) in a murine xenograft model, without any increases in angiogenesis. Similarly, PAI-1/2(+) fibroblasts stimulate experimental metastasis of MDA-MB-231 cells using an in vivo lung colonization assay. Further mechanistic studies revealed that fibroblasts overexpressing PAI-1 or PAI-2 display increased autophagy (“self-eating”) and are sufficient to induce mitochondrial biogenesis/activity in adjacent cancer cells, in co-culture experiments. In xenografts, PAI-1/2(+) fibroblasts significantly reduce the apoptosis of MDA-MB-231 tumor cells. The current study provides further support for the “Autophagic Tumor Stroma Model of Cancer” and identifies a novel “extracellular matrix”-based signaling mechanism, by which a loss of stromal Cav-1 generates a metastatic phenotype. Thus, the secretion and remodeling of extracellular matrix components (such as PAI-1/2) can directly regulate both (1) autophagy in stromal fibroblasts and (2) epithelial tumor cell mitochondrial metabolism.