The Role of the Stroma in Carcinogenesis

This systematic review considers the most recent attitudes and news regarding the influence of the stroma on tumor initiation and progression. It is now widely accepted that tumor stroma plays an active role in carcinogenesis. Many different signaling molecules, ligands and signaling pathways recently have been discovered. This review considers the complexity of interactions between malignant cells and its stroma (cross-talk). The recent advances and better understanding of the tumor-stroma interactions will have important impact on the new and combined therapeutic approaches and modalities.


Introduction
The cancer research has largely been guided by so called a reductionist model which considered the tumor as a disease of cancer cells neglecting all other structures surrounding the tumor cells, named the stroma ().The stroma consists of the following structures: -Extracellular matrix -Blood vessels -Inflammatory cells -Fibroblasts () It is well established that there is a reciprocal (mutual) relation between normal epithelium and its stroma.This relation is necessary for the normal development of the tissues and their morphogenesis ().It is also widely accepted that the tumor formation is caused by accumulation of somatic mutations within epithelial NURIJA BILALOVIĆ ET AL.: THE ROLE OF THE STROMA IN CARCINOGENESIS cells.However, tumor behavior is largely influenced by the stroma factors (microenvironment) ().Molecular studies confirmed significant differences between the stroma of normal tissues, carcinoma in situ and invasive carcinomas.These alterations are already obvious between normal tissue and carcinoma in situ ().The most of these alterations are related to the stromal fibroblasts (, ).Also, perturbations between epithelium and its extracellular matrix can cause cancer development ().We emphasize that genetic mutations within tumor cells can cause very important alterations and activation of the stroma.These changes include: a. Neoangiogenesis (development of new blood vessels) b.Inflammatory response and activation of inflammatory cells (lymphocytes, mast cells and macrophages) c.Different expression of extracellular matrix components d.Increased proliferation of stromal fibroblasts (carcinoma associated fibroblasts) It is important to underline that a cross talk and reciprocal relations between tumor and its stroma are necessary for tumor formation and its progression.Different ligands and signal pathways between tumor and stromal cells are included in these interactions (, ).The communication between tumor cells and stroma is primarily based on a paracrine signals.In vivo studies on mouse pre-cancerous epithelial cells model demonstrated that their exposition to the senescent fibroblasts led to irreversible loss of differentiation, invasion and complete malignant transformation ().Also, the senescent fibroblasts in the culture of normal epithelial breast cells can cause disruption of alveolar architecture, functional differentiation and morphogenesis.One of the key molecules involved in these processes is matrix-metalloproteinase- (MMP-).It is interesting that both senescent and cancer-associated fibroblasts produce the same paracrine molecules (signals, growth factors) which can stimulate adjacent epithelium proliferation ().However there are significant differences between two fibroblast types: the tumor fibroblasts are able to induce epithelial carcinogenesis while the senescent fibroblasts are not able to transform normal (non-transformed) epithelial cells into malignant (, ).These investigations support the idea that senescent cells contribute to age-related pathology including cancer ().

Clinical implications
The impact of the stroma has been discussed in many studies.Unfavorable prognostic value of fibrosis in human tumors has been confirmed in the most of them.
The influences of the stromal factors on overall survival and disease-free survival have been analyzed.One of the most investigated factors is TGF-β and its responding receptors.TGF-β is involved in colorectal cancer pathogenesis since its presence is detected in malignant cells of colorectal mucosa ().The presence of TGF-β is more expressed in low-grade carcinomas and its expression is correlated with overall survival and relapse-free survival.However, TGF-β type  is related to unfavorable prognosis and it is involved in disease progression ().Unlike in colorectal cancer, in head and neck tumors, TGFβ did not show any prognostic value ().In the case of prostate cancer, the higher TGF-β expression is linked to decreased expression of its responding receptors and higher aggressiveness of the prostate cancer.It includes higher Gleason score and more frequent extracapsular invasion of prostate cancer ().CD enzyme, a neutral endopeptidase on surface of stromal fibroblasts, has also been investigated.Higher expression of CD in basal cell carcinoma correlated positively with aggressiveness and invasiveness of the tumor ().Expression of CD in stroma of the breast cancer revealed that CD had been not only the marker of invasiveness but also independent prognostic factor in breast cancer ().
Our study on CD in breast cancer did not confirm these results ().In the case of the gastric cancer, CD expression on the surface of stromal fibroblasts positively correlated with tumor and vascular invasion depth as well as with metastasis to the regional lymph nodes ().The study of Ogawa et al. () also confirmed importance of CD enzyme in invasion and metastasis of colorectal cancer.In malignant melanoma, higher CD expression within both melanoma cells and stromal fibroblasts correlated with invasiveness and metastasis of the tumor cells ().In breast cancer, the presence of stromal fibroblasts generally has been linked to tumor aggressiveness and to prognosticators related to worse outcome ().The study clearly demonstrated that secretory molecules produced by stromal fibroblasts cor-related with aggressiveness and higher metastatic potential of the tumor.Interestingly the investigators found positive correlation between the presence of the stromal fibroblasts and neoangiogenesis.In vitro studies have already demonstrated that stromal fibroblasts induce neoangiogenesis by production of hypoxia induced vascular growth factor (). TGF-β signaling pathways have been involved in the potential therapeutic implications.Specific TGF-β inhibitors have been synthesized.Their application significantly decreased the metastatic potential of the primary breast cancer without important side effects.The novel TGF-β inhibitors also opened a question and dilemma: Did such inhibitors induce the tumor development in normal stroma inhibiting (or blocking) TGF-β?Therefore the clinical use in treatment of human tumors of TGF-β inhibitors has been limited so far.The second important therapeutic implication was related to HGF molecule.Specific HGF inhibitors also have been synthesized.These inhibitors were able to decrease invasiveness of the pancreatic cancer since in vitro studies had revealed high expression of HGF in pancreatic cancer.The stroma of pancreatic cancer was exposed to irradiation and it resulted in several mutations including those related to higher HGF activity.

Conclusion
It is clear today that carcinogenesis is not possible without the active role of the stroma (especially in the case of carcinomas).The mutual relation and the complex signaling pathways (cross-talk) between epithelium and stroma are basic mechanism of malignant alteration and progression.The recent advances revealed the importance of the stromal and senescent fibroblasts and their active role in carcinogenesis.Numerous signaling molecules (especially TGF-β), signaling pathways as well as specific gene mutations have been discovered within the stromal cells.Three-dimensional (D) systems of the cell cultures have been developed to simulate the tumor microenvironment in vivo.These advances enable deeper understanding of the tumor development and treatment modalities.Unfortunately a specific drug has not been developed till now.However research is going on and it probably will result in novel therapeutic approaches based on recent discoveries.