Desmoplastic tumors correspond to a unique tissue structure characterized by the abnormal deposition of extracellular matrix. Breast tumors are a typical example of this type of lesion, a property that allows its palpation and early detection. Fibrillar type I collagen is a major component of tumor desmoplasia and its accumulation is causally linked to tumor cell survival and metastasis. For many years, the desmoplastic phenomenon was considered to be a reaction and response of the host tissue against tumor cells and, accordingly, designated as “desmoplastic reaction”. This notion has been challenged in the last decades when desmoplastic tissue was detected in breast tissue in the absence of tumor. This finding suggests that desmoplasia is a preexisting condition that stimulates the development of a malignant phenotype. With this perspective, in the present review, we analyze the role of extracellular matrix remodeling in the development of the desmoplastic response. Importantly, during the discussion, we also analyze the impact of obesity and cell metabolism as critical drivers of tissue remodeling during the development of desmoplasia. New knowledge derived from the dynamic remodeling of the extracellular matrix may lead to novel targets of interest for early diagnosis or therapy in the context of breast tumors.

Breast tumors belong to the type of desmoplastic lesion in which a stiffer tissue structure is a determinant of breast cancer progression and constitutes a risk factor for breast cancer development. It has been proposed that cancer-associated stromal cells (responsible for this fibrotic phenomenon) are able to metabolize glucose via lactate production, which supports the catabolic metabolism of cancer cells. The aim of this work was to investigate the possible functional link between these two processes. To measure the effect of matrix rigidity on metabolic determinations, we used compliant elastic polyacrylamide gels as a substrate material, to which matrix molecules were covalently linked. We evaluated metabolite transport in stromal cells using two different FRET (Fluorescence Resonance Energy Transfer) nanosensors specific for glucose and lactate. Cell migration/invasion was evaluated using Transwell devices. We show that increased stiffness stimulates lactate production and glucose uptake by mammary fibroblasts. This response was correlated with the expression of stromal glucose transporter Glut1 and monocarboxylate transporters MCT4. Moreover, mammary stromal cells cultured on stiff matrices generated soluble factors that stimulated epithelial breast migration in a stiffness-dependent manner. Using a normal breast stromal cell line, we found that a stiffer extracellular matrix favors the acquisition mechanistical properties that promote metabolic reprograming and also constitute a stimulus for epithelial motility. This new knowledge will help us to better understand the complex relationship between fibrosis, metabolic reprogramming, and cancer malignancy.

Hard consistency, developed under the influence of tumor cell factors, is a characteristic feature of a breast tumor. Activation of resident fibroblasts leading to a myofibroblast phenotype is the principal feature that orchestrates this fibrotic process. The aim of this study was to assess the effects induced by TGF-β1, a growth factor abundantly present in tumor microenvironment, on the molecular mechanisms that mediate myofibroblastic differentiation of normal human mammary fibroblasts. We used an immortalized fibroblastic cell line derived from normal mammary tissue (RMF-EG cells) to study the effect of TGF-β1 in the expression of α-SMA and CTGF as markers of myofibroblastic differentiation. The influence of redox status and JNK activity on TGF-β1-induced transcriptional activity was measured by a luciferase reporter assay. We also used a shRNA approach to evaluate the influence of NOX4 in myofibroblastic differentiation. TGF-β1 stimulates the expression of myofibroblast markers α-SMA and CTGF. Using a NOX inhibitor (DPI) and cells expressing a shRNA for NOX4, we demonstrated that TGF-β1 promotes an oxidative environment that favors myofibroblastic differentiation. We also found that activation of c-Jun N-terminal kinase is required for TGF-β1-dependent expression of CTGF, NOX4 and α-SMA. Human mammary stromal fibrosis, evaluated by the expression of early and late markers as CTGF and α-SMA, depends on the activation of JNK signaling pathway. Our results show that JNK activation is an early event that precedes the increase in ROS levels leading to myofibroblastic differentiation and tumor fibrosis, suggesting that inhibition of JNK may be used a method to interrupt the development of tumor desmoplasia.

It has been proposed that epithelial cells can acquire invasive properties through exposure to paracrine signals originated from mesenchymal cells within the tumor microenvironment. Transforming growth factor-β (TGF-β) has been revealed as an active factor that mediates the epithelial–stroma cross-talk that facilitates cell invasion and metastasis. TGF-β signaling is modulated by the coreceptor Endoglin (Eng), which shows a tumor suppressor activity in epithelial cells and regulates the ALK1-Smad1,5,8 as well as the ALK5-Smad2,3 signaling pathways. In the current work, we present evidence showing that cell surface Eng abundance in epithelial MCF-7 breast cancer cells is inversely related with cell motility. Shedding of Eng in MCF-7 cell surface by soluble matrix metalloproteinase-14 (MMP-14) derived from the HS-5 bone-marrow-derived cell line induces a motile epithelial phenotype. On the other hand, restoration of full-length Eng expression blocks the stromal stimulus on migration. Processing of surface Eng by stromal factors was demonstrated by biotin-neutravidin labeling of cell surface proteins and this processing generated a shift in TGF-β signaling through the activation of Smad2,3 pathway. Stromal MMP-14 abundance was stimulated by TGF-β secreted by MCF-7 cells acting in a paracrine manner. In turn, the stromal proteolytic activity of soluble MMP-14, by inducing Eng shedding, promoted malignant progression. From these data, and due to the capacity of TGF-β to regulate malignancy in epithelial cancer, we propose that stromal-dependent epithelial Eng shedding constitutes a putative mechanism that exerts an environmental control of cell malignancy.

Mitochondrial Complex I plays a crucial role in the proliferation, chemoresistance, and metastasis of breast cancer (BC) cells. This highlights it as an attractive target for anti-cancer drugs. Using submitochondrial particles, we identified FRV–1, an ortho-carbonyl quinone, which inhibits NADH:duroquinone activity in D-active conformation and reduces the 3ADP state respiration dependent on Complex I, causing mitochondrial depolarization, ATP drop, increased superoxide levels, and metabolic remodeling towards glycolysis in BC cells. Introducing methyl groups at FRV–1 structure produced analogs that acted as electron acceptors at the Complex I level or increased the inhibitory effect of FCCP-stimulated oxygen consumption rate, which correlated with their redox potential, but increased toxicity on RMF-621 human breast fibroblasts was observed. FRV–1 was inactive in the naphthoquinone oxidoreductase 1 (NOQ1)-positive BC cell line, MCF7, but the sensitivity was recovered by dicoumarol, a NOQ1 inhibitor, suggesting that FRV–1 is a NOQ1 substrate. Importantly, FRV–1 selectively inhibited the proliferation, migration, and invasion of NQO1 negative BC cell, MDA-MB-231, in an OXPHOS- and ROS-dependent manner and sensitized it to the BH3 mimetic drug venetoclax. Overall, FRV–1 is a novel Complex I inhibitor in D-active conformation, blocking possibly the re-activation to A-state, producing selective anti-cancer effects in NQO1-negative BC cell lines.

From the venom of the Bothrops pictus snake, an endemic species from Peru, we recently have described toxins that inhibited platelet aggregation and cancer cell migration. In this work, we characterize a novel P-III class snake venom metalloproteinase, called pictolysin-III (Pic-III). It is a 62 kDa proteinase that hydrolyzes dimethyl casein, azocasein, gelatin, fibrinogen, and fibrin. The cations Mg²⁺ and Ca²⁺ enhanced its enzymatic activity, whereas Zn²⁺ inhibited it. In addition, EDTA and marimastat were also effective inhibitors. The amino acid sequence deduced from cDNA shows a multidomain structure that includes a proprotein, metalloproteinase, disintegrin-like, and cysteine-rich domains. Additionally, Pic-III reduces the convulxin- and thrombin-stimulated platelet aggregation and in vivo, it has hemorrhagic activity (DHM = 0.3 µg). In epithelial cell lines (MDA-MB-231 and Caco-2) and RMF-621 fibroblast, it triggers morphological changes that are accompanied by a decrease in mitochondrial respiration, glycolysis, and ATP levels, and an increase in NAD(P)H, mitochondrial ROS, and cytokine secretion. Moreover, Pic-III sensitizes to the cytotoxic BH3 mimetic drug ABT-199 (Venetoclax) in MDA-MB-231 cells. To our knowledge, Pic-III is the first SVMP reported with action on mitochondrial bioenergetics and may offer novel opportunities for promising lead compounds that inhibit platelet aggregation or ECM–cancer-cell interactions.