The molecular crosstalk between neoplastic and the surrounding tissue induces several stromal changes, including neoangiogenesis and immune/inflammatory reaction, as well as new extracellular matrix formation and the activation of fibroblast-like cells, a process known as desmoplasia , . Initially, the desmoplastic response was considered a barrier against tumor invasion, but there is growing evidence that desmoplasia is an unfavorable prognostic factor. For example, Sis et al.  suggested that desmoplastia is related to increased risks of regional metastases, poorly differentiated primary tumors and lymphatic and venous invasion in colorectal carcinoma. Similar results were observed for head and neck squamous cell carcinomas, which show a high risk of neck recurrence in presence of a desmoplastic stromal pattern .
In the present study, we investigated the influence of soluble paracrine factors produced in vitro by stromal cells derived from an oral carcinoma and by a neoplastic epithelial cell line on proliferation and gene/protein expression. First, we noted that conditioned medium from stromal fibroblast cultures inhibited Hep-2 cell line proliferation and induced apoptosis, suggesting that factors secreted by fibroblasts include proteins that interfere in cell growth and death of neoplastic cells. In addition, using rapid subtraction hybridization and proteomic analysis, we identified gene products generated by stromal and neoplastic cells that may influence proliferation, differentiation and apoptosis, or drive response to stimulus.
Down-regulated genes in neoplastic cells treated with FCM are involved in signal transduction (FAS, SQSTM1, YWHAZ), transcription (ARID4A, CALR, MYC, PARP1, RNF10, SQSTM1), translation (AARS, RPLP0, RPS17, RPS23), apoptosis (CALR, FAS, TPT1, YWHAZ), cell migration (TMSB4X, GNB2L1), cell cycle and cell proliferation (DYNC1H1, GPNMB, LDOC1, MYC, PSM), epidermis development (UGCG), response to stimulus (EIF2AK1, LTA4H, SQSTM1), transport (CALR, NDUFA4, SQSTM1) and different metabolic processes (USP9X). Up-regulated genes are also involved in transcription and translation (ENO1, EIF1, TARS), apoptosis (DAP3, RTN3), cell proliferation (PRDX1, ENO1), organ development (PRDX1), response to stress (EIF1, RTN3) and metabolic processes (PRDX1, P4HB, USP39).
In fibroblasts treated with HCM, the biological processes of down-regulated genes include signal transduction (S100A6, FN1), transcription and translation (FOSL1, RPL37A, RPL7, RPL19, RPL27A, RPLP0), apoptosis (CTSB, TPT1), cell proliferation (S100A6, FOSL1), epidermis development (COL1A1), response to stimulus (FN1, FOSL1), transport (ERGIC3, STX4) and protein and RNA metabolism (CTSB, PRPF3).
Two genes exhibited similar patterns in both cells (RPLP0, TPT1), which may indicate that the transcript levels are affected by soluble paracrine factors produced by either fibroblasts or neoplastic cells or by other in vitro conditions. Therefore, they may not be specific to interactions between stroma and tumor.
After literature analysis, nine genes (ARID4A, CALR, GNB2L1, GPNMB, RNF10, SQSTM1, USP9X, PRDX1 and DAP3) showing potential involvement in signaling cascades related to tumorigenesis and/or stromal/tumor cell interactions were selected for validation by real-time RT-PCR using treated and non-treated cell lines. For six genes (ARID4A, CALR, GNB2L1, RNF10, SQSTM1, USP9X), the results were consistent with the RASH data. In almost half of the primary tumors analyzed, ARID4A transcripts also showed down-regulation, although no correlation with clinicopathological features was detected. These findings in primary tumors should reflect the complex network of a multi-cellular tissue, a situation contrasting with that of a neoplastic cell line cultured in medium conditioned by fibroblasts.
The product of ARID4A - AT rich interactive domain 4A (RBP1-like) - also known as RBP1 or RBBP1 gene, interacts with the tumor suppressor retinoblastoma (pRB) and histone-modifying complexes, repressing promoters of specific genes . Röhl et al.  detected several genes, including ARID4A, overexpressed in astrocytes treated with medium conditioned by activated microglia, which protected them against stress conditions. Recently, Wu et al.  showed that Arid4a-deficient mice exhibit down-regulation of several homeobox genes and of the forkhead box gene Foxp3, which codes a transcription factor involved in the development and function of regulatory T cells . These mice also show bone marrow failure with myelofibrosis and higher frequencies of hematologic malignancies, providing evidence that ARID4A functions as a tumor suppressor gene and its absence is permissive for the proliferation of connective tissue elements. The study of Perez et al.  added data on the role of this gene in cancer. These authors detected increased mRNA levels of ARID4A and RB1 in normal human epidermal keratinocytes treated with arsenic and benzo [a]pyrene in vitro. Since these chemicals alter proliferation and inhibit differentiation of keratinocytes [63–65], the findings may indicate that up-regulation of ARID4A is negatively related to epithelial differentiation. Therefore, the potential modulation of this gene by paracrine factors produced by stromal fibroblasts may represent an attempt to promote differentiation of neoplastic epithelial cells and, at the same time, their proliferation.
Calreticulin (coded by CALR or CRT gene) is a calcium-binding protein of the endoplasmic reticulum with intracellular and extracellular functions related to cellular adhesion, migration, and phagocytosis . Calreticulin can be observed on the surface of stressed cells and, when bound to the plasma membrane of apoptotic cells, drives the phagocytosis by macrophages and dendritic cells . In absence of this protein, the cells are not efficiently removed by phagocytes . Recently, Nanney et al.  showed that calreticulin stimulates both migration and proliferation of keratinocytes and fibroblasts and apparently attracts monocytes and macrophages, suggesting its involvement in inflammatory response. Otherwise, fibroblasts underexpressing CARL exhibit weak adhesion and spreading . Accordingly, Kypreou et al.  detected a correlation between calreticulin up-regulation and progression of fibrosis and also that TGF-beta, a contributing factor in fibrotic processes, up-regulated calreticulin in cultured human epithelial cells. In light of the data, we speculate that the low levels of this protein observed in treated Hep-2 cells inhibit proliferation, or represent a protective response of neoplastic cells to phagocytosis and antitumor immune process.
Guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1 or Rack1 (coded by GNB2L1 gene) is a cytosolic protein homologous to the beta subunit of G proteins, and contains seven WD repeats, which act as sites for protein-protein interactions. Binding partners of GNB2L1 include protein kinase C, Src family kinases, components of the ERK pathway, cytokine and interferon receptors, beta integrins and many others. Many of these interactions are consistent with the participation of Rack1 in cell adhesion, movement and growth [72–75].
Sequestosome 1 or ubiquitin-binding protein p62 (coded by SQSTM1 or p60 or p62 gene) is a 62-kDa protein that binds to the Src homology 2 (SH2) domain of p56lck kinase in a phosphotyrosine-independent manner . It has been suggested that p62 is a signaling adaptor which links different signal transduction pathways related to cell proliferation, differentiation and death, including NF-κB pathway [77–82]. SQSTM1 abnormal expression has been observed in hepatocellular, prostate and breast cancers [83–85] and is associated with poor outcomes in breast cancer .
Another gene down-regulated by fibroblast-conditioned medium is USP9X (ubiquitin specific peptidase 9, X-linked), also known as DFFRX, FAF or FAM. This gene is a member of the peptidase C19 family and encodes a protein similar to ubiquitin-specific proteases (USPs). These proteases regulate the production and recycling of ubiquitin and are critically involved in the control of cell growth, differentiation, and apoptosis . Alteration of USPs may play an important role in the pathogenesis of cancer  and may exert distinct growth regulatory activities by acting as oncoproteins or tumor suppressor proteins. Overexpression of certain USPs correlates with progression towards a more malignant phenotype in carcinoma of lung, kidney, breast and prostate [89, 90].
RNF10 (ring finger protein 10) is the least known gene selected for validation. The product contains a ring finger motif, which is involved in protein-protein interactions and has been described in proteins implicated in many cellular processes such as signal transduction, transcriptional regulation, ubiquination, and apoptosis [91, 92].
With respect to proteomic analysis, few differences (mostly quantitative) between treated and non-treated cells were detected. Among the proteins differentially expressed, alpha-enolase, heterogeneous nuclear ribonucleoprotein C C1/C2, aldolase A, tubulin beta and glyceraldehyde-3-phosphate dehydrogenase were down-regulated in neoplastic cells treated with FCM and vimentin and actin were down-regulated in fibroblasts treated with HCM. These proteins, produced by neoplastic cells or fibroblasts, may affect tumorigenesis. For example, the glycolytic enzyme alpha-enolase and its enzymatically inactive isoform MBP-1 (c-myc promoter binding protein 1) are negative regulators for MYC expression [93, 94]. MYC is one of the most frequently de-regulated oncogenes in cancer  and, in the absence of both enzymes, may become activated and accelerate tumor growth. Contrary to RaSH results, alpha enolase protein was observed underexpressed by proteomic analysis in treated Hep-2 cells, which may indicate a nonspecific finding or a post-transcriptional/posttranslational regulation of the RNA/enzyme.