Roberto Lugo

Aberrant DNA methylation in non-small cell lung cancer-associated fibroblasts

Summary DNA methylation profiling of TAFs reveals global demethylation and a selective impact on the TGF-β pathway. Moreover, it suggests the fibrocyte origin of a fraction of TAFs, and identifies a novel prognostic biomarker in non-small cell lung cancer.

Matrix Stiffening and β1 Integrin Drive Subtype-Specific Fibroblast Accumulation in Lung Cancer

The crucial role of tumor-associated fibroblasts (TAF) in cancer progression is now clear in non–small cell lung cancer (NSCLC). However, therapies against TAFs are limited due to a lack of understanding in the subtype-specific mechanisms underlying their accumulation. Here, the mechanical (i.e., matrix rigidity) and soluble mitogenic cues that drive the accumulation of TAFs from major NSCLC subtypes: adenocarcinoma (ADC) and squamous cell carcinoma (SCC) were dissected. Fibroblasts were cultured on substrata engineered to exhibit normal- or tumor-like stiffnesses at different serum concentrations, and critical regulatory processes were elucidated. In control fibroblasts from nonmalignant tissue, matrix stiffening alone increased fibroblast accumulation, and this mechanical effect was dominant or comparable with that of soluble growth factors up to 0.5% serum. The stimulatory cues of matrix rigidity were driven by β1 integrin mechano-sensing through FAK (pY397), and were associated with a posttranscriptionally driven rise in β1 integrin expression. The latter mechano-regulatory circuit was also observed in TAFs but in a subtype-specific fashion, because SCC–TAFs exhibited higher FAK (pY397), β1 expression, and ERK1/2 (pT202/Y204) than ADC–TAFs. Moreover, matrix stiffening induced a larger TAF accumulation in SCC–TAFs (>50%) compared with ADC–TAFs (10%–20%). In contrast, SCC–TAFs were largely serum desensitized, whereas ADC–TAFs responded to high serum concentration only. These findings provide the first evidence of subtype-specific regulation of NSCLC–TAF accumulation. Furthermore, these data support that therapies aiming to restore normal lung elasticity and/or β1 integrin-dependent mechano regulation may be effective against SCC–TAFs, whereas inhibiting stromal growth factor signaling may be effective against ADC–TAFs. Implications: This study reveals distinct mechanisms underlying the abnormal accumulation of tumor-supporting fibroblasts in two major subtypes of lung cancer, which will assist the development of personalized therapies against these cells. Mol Cancer Res; 13(1); 161–73. ©2014 AACR.

Heterotypic paracrine signaling drives fibroblast senescence and tumor progression of large cell carcinoma of the lung

Senescence in cancer cells acts as a tumor suppressor, whereas in fibroblasts enhances tumor growth. Senescence has been reported in tumor associated fibroblasts (TAFs) from a growing list of cancer subtypes. However, the presence of senescent TAFs in lung cancer remains undefined. We examined senescence in TAFs from primary lung cancer and paired control fibroblasts from unaffected tissue in three major histologic subtypes: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Three independent senescence markers (senescence-associated beta-galactosidase, permanent growth arrest and spreading) were consistently observed in cultured LCC-TAFs only, revealing a selective premature senescence. Intriguingly, SCC-TAFs exhibited a poor growth response in the absence of senescence markers, indicating a dysfunctional phenotype rather than senescence. Co-culturing normal fibroblasts with LCC (but not ADC or SCC) cancer cells was sufficient to render fibroblasts senescent through oxidative stress, indicating that senescence in LCC-TAFs is driven by heterotypic signaling. In addition, senescent fibroblasts provided selective growth and invasive advantages to LCC cells in culture compared to normal fibroblasts. Likewise, senescent fibroblasts enhanced tumor growth and lung dissemination of tumor cells when co-injected with LCC cells in nude mice beyond the effects induced by control fibroblasts. These results define the subtype-specific aberrant phenotypes of lung TAFs, thereby challenging the common assumption that lung TAFs are a heterogeneous myofibroblast-like cell population regardless of their subtype. Importantly, because LCC often distinguishes itself in the clinic by its aggressive nature, we argue that senescent TAFs may contribute to the selective aggressive behavior of LCC tumors.

Epithelial contribution to the profibrotic stiff microenvironment and myofibroblast population in lung fibrosis

Myofibroblasts are key effector cells in tissue stiffening and fibrosis progression, but the contribution of cells undergoing epithelial–mesenchymal transition (EMT) is unclear. Unlike EMT, myofibroblasts contribute to tissue stiffening through their contractility and expression of fibrillar collagens, which is associated with aberrant FAK (Y397) hyperactivation.

Abstract 4103: Cancer cell-stromal cell crosstalk drives fibroblast senescence and tumor progression in large cell carcinoma of the lung in culture and in vivo

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Permanent cell cycle arrest through senescence has been previously regarded as a protective mechanism against tumor progression. In contrast, there is evidence that senescence in tumor-associated fibroblasts (TAFs) enhances tumor growth. Senescence has been reported in tumor associated fibroblasts (TAFs) from a growing list of selected cancer types. However, the presence of senescent TAFs in lung cancer remains undefined. To address this gap of knowledge, we examined common markers of senescence in primary TAFs from the 3 major non-small cell lung cancer (NSCLC) subtypes: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Given the difficulties in gathering LCC-TAFs owing to the lower prevalence of LCC compared to the other subtypes, primary fibroblasts from 2 independent cell collections were used. We found an enrichment of the myofibroblast-like phenotype in TAFs regardless their histologic subtype, yet senescence was observed in LCC-TAFs only. Likewise, co-culturing normal lung fibroblasts with LCC (but not ADC or SCC) cancer cells was sufficient to induce senescence, and this induction was prevented in the presence of an antioxidant, indicating that it is mediated through oxidative stress. Of note, senescent fibroblasts provided growth and invasive advantages to LCC cells in culture and in vivo beyond those provided by control (non-senescent) fibroblasts. These results expand recent evidence that challenges the common assumption that lung TAFs are a heterogeneous myofibroblast-like cell population regardless their histologic subtype. Of note, because LCC often distinguishes itself in the clinic by its aggressive nature, our findings support that senescent or senescent-like TAFs may contribute to the selective aggressive behavior of LCC tumors. Citation Format: Roberto Lugo, Marta Gabasa, Francesca Andriani, Marta Puig, Federica Facchinetti, Josep Ramirez, Abel Gomez-Caro, Ugo Pastorino, Pere Gascon, Albert Davalos, Noemi Reguart, Luca Roz, Jordi Alcaraz. Cancer cell-stromal cell crosstalk drives fibroblast senescence and tumor progression in large cell carcinoma of the lung in culture and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4103.

Abstract 1089: Matrix stiffening and β1 integrin promote fibroblast accumulation in lung squamous cell carcinomas but not in adenocarcinomas

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The mechanisms underlying the abundance of tumor associated fibrobasts (TAFs) in non-small cell lung cancer (NSCLC) remain poorly understood. To address this gap of knowedge, we examined the relative contribution of mechanical (i.e. matrix rigidity) and soluble biochemical (i.e. serum) cues in the accumulation of primary TAFs from either adenocarcinoma (ADC) or squamous cell carcinoma (SQC) NSCLC patients. For this purpose, fibroblasts were cultured on collagen-I coated gels engineered to exhibit either normal- or tumor-like stiffnesses at different serum concentrations. In fibroblasts from non-malignant tissue, matrix stiffening alone was sufficient to increase fibroblast accumulation by 20%. In addition, matrix stiffening-induced accumulation was dominant or comparable to that due to soluble growth factors up to moderate serum concentrations. These mechanical stimulatory effects were β1 integrin-dependent, and were associated with both increased integrin mechanosensing through FAKpY397and a post-transcriptionally-driven rise in β1 integrin expression. The latter mechanoregulatory circuit could be extended to TAFs in a subtype-dependent fashion, since SQC-TAFs exhibited higher FAKpY397 and β1 expression than ADC-TAFs. Remarkably, SQC-TAF density was enhanced > 50% in response to matrix stiffening, whereas they were largely desensitized to serum. Conversely, substantial accumulation of ADC-TAFs required large serum concentration, whereas it was poorly induced by either matrix stiffening or low serum concentration. These results reveal that a therapeutic strategy aiming to inhibit soluble growth factors may be effective against ADC-TAFs, whereas a strategy aiming to rescue normal lung elasticity may be more efficient against SQC-TAFs. Citation Format: Jordi Alcaraz, Marta Puig, Roberto Lugo, Alicia Gimenez, Adriana Velasquez, Roland Galgoczy, Josep Ramirez, Abel Gomez-Caro, Pere Gascon, Noemi Reguart. Matrix stiffening and β1 integrin promote fibroblast accumulation in lung squamous cell carcinomas but not in adenocarcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1089. doi:10.1158/1538-7445.AM2014-1089

Abstract 1103: An abnormally stiff microenvironment supports the overabundance of fibroblasts in non-small cell lung cancer.

Breathing demands our lungs to be soft and elastic. In contrast, lung tumors exhibit an abnormal tissue hardening concomitantly with an altered lung architecture and function, which brings the mechanical microenvironment of the tumor closer to that of muscle or bone. This tissue hardening has been associated with the abundant reactive stroma rich in activated fibroblasts (also referred to as tumor associated fibroblasts or TAFs) that is a hallmark of non-small cell lung cancer (NSCLC). However, it remains unknown whether tissue hardening alone is sufficient to drive the abnormal abundance of fibroblasts in the stroma of NSCLC. To address this question, we cultured the human lung fibroblast cell line CCD-19Lu or primary human lung fibroblasts on collagen-coated polyacrylamide gels exhibiting normal- or tumor-like stiffness in the absence (0%) or presence (0.1%) of serum for 5 days. Primary cells were isolated from both tumor-free regions (referred to as control fibroblasts) or tumors of NSCLC patients diagnosed with either Adenocarcinoma (ADC, n=5) or Squamous Cell Carcinoma (SCC, n=5) histological subtypes. We observed a significantly higher density of CCD-19Lu fibroblasts in tumor-like gels compared to normal-like gels regardless the presence of serum. Similar results were obtained in both primary control fibroblasts and SCC-TAFs. In contrast, tumor-like gels induced a weaker density increase in ADC-TAFs. The increased fibroblast density in the tumor-like gels was associated with increased survival rather than proliferation, as revealed by a downregulation of caspase-3 and a rise in AKT phosphorylation. Our results indicate that extracellular hardening alone is sufficient to induce an increased density in lung fibroblasts by activating pro-survival pathways and suggest an abnormal response of ADC-TAFs to extracellular mechanical cues. Collectively these findings underline a major role of tissue hardening in the abnormal abundance of TAFs in NSCLC, particularly in the SCC subtype. These data may be useful in developing novel targeted therapies against the pro-survival effects of tissue hardening in SCC-TAFs, which act as co-conspirators of tumor progression. Citation Format: Marta Puig, Alicia Gimenez, Roland Galgoczy, Roberto Lugo, Josep Ramirez, Abel Gomez-Caro, Pere Gascon, Noemi Reguart, Jordi Alcaraz. An abnormally stiff microenvironment supports the overabundance of fibroblasts in non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1103. doi:10.1158/1538-7445.AM2013-1103