Collagen type I induces EGFR-TKI resistance in EGFR-mutated cancer cells by mTOR activation through Akt-independent pathway.

Primary resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a serious problem in lung adenocarcinoma patients harboring EGFR mutations. The aim of this study was to examine whether and how collagen type I (Col I), the most abundantly deposited matrix in tumor stroma, affects EGFR-TKI sensitivity in EGFR-mutant cells. We evaluated the EGFR-TKI sensitivity of EGFR-mutated cancer cells cultured with Col I. Changes in the activation of downstream signaling molecules of EGFR were analyzed. We also examined the association between the Col I expression in tumor stroma in surgical specimens and EGFR-TKI response of postoperative recurrence patients with EGFR mutations. Compared to cancer cells without Col I, the survival rate of cancer cells cultured with Col I was significantly higher after EGFR-TKI treatment. In cancer cells cultured with and without Col I, EGFR-TKI suppressed the levels of phosphorylated (p-)EGFR, p-ERK1/2, and p-Akt. When compared to cancer cells without Col I, expression of p-P70S6K, a hallmark of mTOR activation, was dramatically upregulated in cancer cells with Col I. This activation was maintained even after EGFR-TKI treatment. Simultaneous treatment with EGFR-TKI and mTOR inhibitor abrogated Col I-induced resistance to EGFR-TKI. Patients with Col I-rich stroma had a significantly shorter progression-free survival time after EGFR-TKI therapy (238 days vs 404 days; P < .05). Collagen type I induces mTOR activation through an Akt-independent pathway, which results in EGFR-TKI resistance. Combination therapy using EGFR-TKI and mTOR inhibitor could be a possible strategy to combat this resistance.

Site-specific fibroblasts regulate site-specific inflammatory niche formation in gastric cancer.

BACKGROUND: Fibroblasts are the commonest type of cancer stromal cells. Inflammation occurs in cancer tissue, and the inflammatory process has been suggested to be caused by interactions between immune cells and cancer cells. In this study, we clarified that site-specific fibroblasts regulate the formation of a site-specific inflammatory niche according to the depth of gastric cancer cell invasion. METHODS: Immunohistochemistry was performed with paraffin-embedded tissues. The numbers of immune cells and the fibroblast area were calculated according to the cancer depth. The gene expression patterns of submucosal fibroblasts and subperitoneal fibroblasts stimulated with HSC44PE-conditioned medium were analyzed with a microarray. To examine the effects on the cancer microenvironment of differences in gene expressions between HSC44PE-stimulated submucosal fibroblasts and subperitoneal fibroblasts, assays of HSC44PE proliferation, T cell migration, and M2-like macrophage differentiation were performed. RESULTS: The distributions of immune cells differed between the submucosal layer and the subserosal layer. The number of M2 macrophages was significantly higher and the fibroblast area was significantly larger in the subserosal layer compared with the submucosal layer. High expression levels of IL1B, TNFSF15, and CCL13 were observed in HSC44PE-stimulated submucosal fibroblasts, and higher expression levels of TGFB2, CSF1, CCL8, and CXCL5 were found in HSC44PE-stimulated subperitoneal fibroblasts. HSC44PE-stimulated subperitoneal fibroblast medium promoted the differentiation of monocytes into M2-like macrophages, whereas HSC44PE-stimulated submucosal fibroblasts significantly induced the migration of Jurkat cells and the growth of HSC44PE cells. CONCLUSION: The dynamic states of immune cells differ between the submucosal and subserosal layers in cancer tissues. Site-specific fibroblasts regulate site-specific inflammatory niche formation according to the depth of cancer cell invasion.

Gene expression profile in the activation of subperitoneal fibroblasts reflects prognosis of patients with colon cancer.

Tumors can create a heterogenetic tumor microenvironment. We recently identified the pathologically unique cancer microenvironment formed by peritoneal invasion (CMPI), and revealed that subperitoneal fibroblasts (SPFs) within peritoneal tissue play a crucial role in tumor progression through their interaction with cancer cells. Therefore, the genes in SPFs altered by cancer stimulation may include some biologically important factors associated with patient prognosis. In this study, we aimed to identify new biomarkers using genes specifically upregulated in SPFs by cancer-cell-conditioned medium (CCCM) stimulation (SPFs CCCM response genes; SCR genes) in colon cancer (CC). We constructed two frameworks using SCR gene data: a publicly released microarray dataset, and validation cases with freshly frozen CC samples to identify genes related to short recurrence-free survival (RFS). In the first framework, we selected differentially expressed genes between the high and low SCR gene expression groups. In the second framework, genes significantly related to short RFS were selected by univariate analysis using all SCR genes, and multivariate analysis was performed to select robust genes associated with short RFS. We identified CTGF, CALD1, INHBA and TAGLN in the first framework, and PDLIM5, MAGI1, SPTBN1 and TAGLN in the second framework. Among these seven genes, high expression of three genes (CALD1, TAGLN and SPTBN1) showed a poor prognosis in our validation cases. In a public microarray dataset, SCR gene expression was associated with the expression of ECM component, EMT, and M2-macrophage associated genes, which was concordant with the pathological features of CMPI. Thus, we successfully identified new prognostic factors.

Spread of tumor microenvironment contributes to colonic obstruction through subperitoneal fibroblast activation in colon cancer.

We evaluated the influence of the cancer microenvironment formed by peritoneal invasion (CMPI) on clinical findings in colon cancer patients. In addition to the association with poor prognosis, we discovered a relationship with bowel obstruction. Detailed analysis revealed that clinical findings related to bowel obstruction occurred more frequently in patients with an elevated type tumor, which had peritoneal elastic laminal elevation to the tumor surface, compared to those with non-elevated type tumors among those with elastic laminal invasion (ELI). Lateral tumor spread and increase of tumor annularity rate in ELI-positive elevated type cases suggested the morphological progression from ELI-positive non-elevated type to elevated type. In addition, α-smooth muscle actin expression was the highest in ELI-positive elevated type, and prominent expressions were found not only in the deep tumor area but also in the shallow tumor area. Furthermore, contraction assays revealed the robust contractile ability of subperitoneal fibroblasts stimulated by cancer cell-conditioned medium. Our findings suggest that CMPI spread into the luminal side of the colonic wall along with tumor progression, which caused bowel obstruction through the activation of subperitoneal fibroblasts. However, although the clinical outcome was not different between the two types, the clinical findings were affected by the spread of CMPI. We are the first to explore how the alteration of the tumor-promoting microenvironment, along with tumor progression, contributes to the development of clinical findings.

High Mobility Group Box1 (HMGB1) released from cancer cells induces the expression of pro-inflammatory cytokines in peritoneal fibroblasts.

High Mobility Group Box1 protein (HMGB1), one of the mediators of inflammation, is associated with tumorigenesis. The HMGB1-Receptor for advanced glycation end-products (RAGE) in gastric adenocarcinoma tissues promoted gastric cancer growth, however, there are no reports concerning the relationship between the expression of HMGB1 in gastric cancer and cancer-related inflammation. Fibroblasts exist most abundantly on cancer tissue where inflammation occurs. So, we studied the effects of HMGB1 released from cancer cells on the fibroblasts. The expression of HMGB1 in cancer cells and nuclear factor-kappa B (NF-kB) in fibroblasts were evaluated immunohistochemically in human gastric cancer specimens. Cytoplasmic HMGB1 expression in the cancer cells and nuclear translocation of NF-kB in fibroblasts were detected at deeper invasion. To determine whether HMGB1 released from cancer cells induces the expression of pro-inflammatory cytokines in fibroblasts, we analyzed the activation of Toll-like receptor (TLR) signaling. Fibroblasts stimulated by recombinant HMGB1 and the HSC44PE-conditioned medium showed the phosphorylation of Interleukin-1 receptor associated-kinase 4 (IRAK4), nuclear translocation of NF-kB, and enhanced pro-inflammatory cytokine expression. Treatment with HSC44PE-conditioned-medium transfected with siRNA-HMGB1 reduced the expressions of pro-inflammatory cytokines in the fibroblasts. We propose that HMGB1 released from cancer cells induces the expression of pro-inflammatory cytokines in peritoneal fibroblasts through the HMGB1-TLR2/4 pathway.

PTPRZ1 regulates calmodulin phosphorylation and tumor progression in small-cell lung carcinoma.

BACKGROUND: Small-cell lung carcinoma (SCLC) is a neuroendocrine tumor subtype and comprises approximately 15% of lung cancers. Because SCLC is still a disease with a poor prognosis and limited treatment options, there is an urgent need to develop targeted molecular agents for this disease. METHODS: We screened 20 cell lines from a variety of pathological phenotypes established from different organs by RT-PCR. Paraffin-embedded tissue from 252 primary tumors was examined for PTPRZ1 expression using immunohistochemistry. shRNA mediated PTPRZ1 down-regulation was used to study impact on tyrosine phosphorylation and in vivo tumor progression in SCLC cell lines. RESULTS: Here we show that PTPRZ1, a member of the protein tyrosine- phosphatase receptor (PTPR) family, is highly expressed in SCLC cell lines and specifically exists in human neuroendocrine tumor (NET) tissues. We also demonstrate that binding of the ligand of PTPRZ1, pleiotrophin (PTN), activates the PTN/PTPRZ1 signaling pathway to induce tyrosine phosphorylation of calmodulin (CaM) in SCLC cells, suggesting that PTPRZ1 is a regulator of tyrosine phosphorylation in SCLC cells. Furthermore, we found that PTPRZ1 actually has an important oncogenic role in tumor progression in the murine xenograft model. CONCLUSION: PTPRZ1 was highly expressed in human NET tissues and PTPRZ1 is an oncogenic tyrosine phosphatase in SCLCs. These results imply that a new signaling pathway involving PTPRZ1 could be a feasible target for treatment of NETs.

Single cell time-lapse analysis reveals that podoplanin enhances cell survival and colony formation capacity of squamous cell carcinoma cells.

Tumor initiating cells (TICs) are characterized by high clonal expansion capacity. We previously reported that podoplanin is a TIC-specific marker for the human squamous cell carcinoma cell line A431. The aim of this study is to explore the molecular mechanism underlying the high clonal expansion potential of podoplanin-positive A431cells using Fucci imaging. Single podoplanin-positive cells created large colonies at a significantly higher frequency than single podoplanin-negative cells, whereas no difference was observed between the two types of cells with respect to cell cycle status. Conversely, the cell death ratio of progenies derived from podoplanin-positive single cell was significantly lower than that of cells derived from podoplanin-negative cells. Single A431 cells, whose podoplanin expression was suppressed by RNA interference, exhibited increased cell death ratios and decreased frequency of large colony forming. Moreover, the frequency of large colony forming decreased significantly when podoplanin-positive single cells was treated with a ROCK (Rho-associated coiled-coil kinase) inhibitor, whereas no difference was observed in single podoplanin-negative cells. Our current study cleared that high clonal expansion capacity of podoplanin-positive TICs populations was the result of reduced cell death by podoplanin-mediated signaling. Therefore, podoplanin activity may be a therapeutic target in the treatment of squamous cell carcinomas.

Gene expression profiling to predict recurrence of advanced squamous cell carcinoma of the tongue: discovery and external validation.

OBJECTIVES: To establish a prognostic signature for locally advanced tongue squamous cell carcinoma (TSCC) patients treated with surgery. RESULTS: In the discovery study, unsupervised hierarchical clustering analysis identified two clusters which differentiated the Kaplan-Meier curves of RFS [median RFS, 111 days vs. not reached; log-rank test, P = 0.023]. The 30 genes identified were combined into a dichotomous PI. In the validation cohort, classification according to the PI was associated with RFS [median RFS, 754 days vs. not reached; log-rank test, P = 0.026 in GSE31056] and DSS [median DSS, 540 days vs. not reached; log-rank test, P = 0.046 in GSE42743 and 443 days vs. not reached; P < 0.001 in GSE41613]. Among genes, positive immunohistochemical staining of cytokeratin 4 was associated with favorable prognostic values for RFS (hazard ratio (HR), 0.591, P = 0.045) and DSS (HR, 0.333, P = 0.004). MATERIALS AND METHODS: We conducted gene expression profiling of 26 clinicopathologically homogeneous advanced TSCC tissue samples using cDNA microarray as a discovery study. Candidate genes were screened using clustering analysis and univariate Cox regression analysis for relapse-free survival (RFS). These were combined into a prognostic index (PI), which was validated using three public microarray datasets of tongue and oral cancer (123 patients). Some genes identified in discovery were immunohistochemically examined for protein expression in another 127 TSCC patients. CONCLUSION: We identified robust molecular markers that showed significant associations with prognosis in TSCC patients. Gene expression profiling data were successfully converted to protein expression profiling data.

Clonal heterogeneity in osteogenic potential of lung cancer-associated fibroblasts: promotional effect of osteogenic progenitor cells on cancer cell migration.

BACKGROUND: Cancer-associated fibroblasts (CAFs) consist of heterogeneous cell population in terms of their differentiation potential. The functional differences in tumor progression between CAFs with mesenchymal stem/progenitor cells (MSCs/MPCs) characteristics and CAFs without MSCs/MPCs characteristics are not clarified. METHODS: CAFs and vascular adventitial fibroblasts (VAFs, which contain MSCs/MPCs) were isolated from nine primary lung cancers and were cultured in osteogenic or adipogenic medium to assess their multi-lineage differentiation. Next, we established nine single-cell-derived clones from the primary culture of CAFs and examined their differentiation potential. The effects of each single-cell-derived clone on the proliferation and migration of lung adenocarcinoma cell line, A549, were analyzed. RESULTS: The nine samples of VAFs and CAFs showed various degrees of osteogenic differentiation. Although the VAFs displayed the ability to undergo adipogenic differentiation, all cases of the CAFs did not. CAFs clones presented varying degrees of osteogenic differentiation. Four clones displayed comparable levels of osteogenic potential with that of the VAFs, and two clones were completely negative. As compared to the CAFs clones that possessed lower osteogenic potential, CAFs clones with higher osteogenic potential did not confer proliferative activity in A549 cells. On the contrary, these clones significantly promoted the migration of A549 cells as compared to the clones with lower osteogenic potential. CONCLUSION: Our studies clearly indicate that CAFs derived from lung cancer are heterogeneous population that consists of cells with varying osteogenic potentials and that CAFs with higher osteogenic potential have a greater tumor-promoting function through the enhancement of cancer cell migration.

Gastrointestinal Fibroblasts Have Specialized, Diverse Transcriptional Phenotypes: A Comprehensive Gene Expression Analysis of Human Fibroblasts.

BACKGROUND: Fibroblasts are the principal stromal cells that exist in whole organs and play vital roles in many biological processes. Although the functional diversity of fibroblasts has been estimated, a comprehensive analysis of fibroblasts from the whole body has not been performed and their transcriptional diversity has not been sufficiently explored. The aim of this study was to elucidate the transcriptional diversity of human fibroblasts within the whole body. METHODS: Global gene expression analysis was performed on 63 human primary fibroblasts from 13 organs. Of these, 32 fibroblasts from gastrointestinal organs (gastrointestinal fibroblasts: GIFs) were obtained from a pair of 2 anatomical sites: the submucosal layer (submucosal fibroblasts: SMFs) and the subperitoneal layer (subperitoneal fibroblasts: SPFs). Using hierarchical clustering analysis, we elucidated identifiable subgroups of fibroblasts and analyzed the transcriptional character of each subgroup. RESULTS: In unsupervised clustering, 2 major clusters that separate GIFs and non-GIFs were observed. Organ- and anatomical site-dependent clusters within GIFs were also observed. The signature genes that discriminated GIFs from non-GIFs, SMFs from SPFs, and the fibroblasts of one organ from another organ consisted of genes associated with transcriptional regulation, signaling ligands, and extracellular matrix remodeling. CONCLUSIONS: GIFs are characteristic fibroblasts with specific gene expressions from transcriptional regulation, signaling ligands, and extracellular matrix remodeling related genes. In addition, the anatomical site- and organ-dependent diversity of GIFs was also discovered. These features of GIFs contribute to their specific physiological function and homeostatic maintenance, and create a functional diversity of the gastrointestinal tract.

TEM1 expression in cancer-associated fibroblasts is correlated with a poor prognosis in patients with gastric cancer.

The cancer stroma, including cancer-associated fibroblasts (CAFs), is known to contribute to cancer cell progression and metastasis, suggesting that functional proteins expressed specifically in CAFs might be candidate molecular targets for cancer treatment. The purpose of the present study was to explore the possibility of using TEM1 (tumor endothelial marker 1), which is known to be expressed in several types of mesenchymal cells, as a molecular target by examining the impact of TEM1 expression on clinicopathological factors in gastric cancer patients. A total of 945 consecutive patients with gastric cancer who underwent surgery at the National Cancer Center Hospital East between January 2003 and July 2007 were examined using a tissue microarray approach. TEM1 expression in CAFs or vessel-associated cells was determined using immunohistochemical staining. Three items (CAF-TEM1-positivity, CAF-TEM1-intensity, and vessel-TEM1-intensity) were then examined to determine the correlations between the TEM1 expression status and the recurrence-free survival (RFS), overall survival (OS), cancer-related survival (COS), and other clinicopathological factors. Significant correlations between CAF-TEM1-positivity or CAF-TEM1-intensity and RFS, OS, or COS were observed (P < 0.001, Kaplan-Meier curves); however, no significant correlation between vessel-TEM1-intensity and RFS, OS, or COS was observed. A univariate analysis showed that CAF-TEM1-positivity and CAF-TEM1-intensity were each correlated with a scirrhous subtype, tumor depth, nodal status, distant metastasis, serosal invasion, lymphatic or venous vessel infiltrations, and pTMN stage. This study suggests that the inhibition of TEM1 expression specifically in the CAFs of gastric carcinoma might represent a new strategy for the treatment of gastric cancer.

Human subperitoneal fibroblast and cancer cell interaction creates microenvironment that enhances tumor progression and metastasis.

BACKGROUNDS: Peritoneal invasion in colon cancer is an important prognostic factor. Peritoneal invasion can be objectively identified as periotoneal elastic laminal invasion (ELI) by using elastica stain, and the cancer microenvironment formed by the peritoneal invasion (CMPI) can also be observed. Cases with ELI more frequently show distant metastasis and recurrence. Therefore, CMPI may represent a particular milieu that facilitates tumor progression. Pathological and biological investigations into CMPI may shed light on this possibly distinctive cancer microenvironment. METHODS: We analyzed area-specific tissue microarrays to determine the pathological features of CMPI, and propagated subperitoneal fibroblasts (SPFs) and submucosal fibroblasts (SMFs) from human colonic tissue. Biological characteristics and results of gene expression profile analyses were compared to better understand the peritoneal invasion of colon cancer and how this may form a special microenvironment through the interaction with SPFs. Mouse xenograft tumors, derived by co-injection of cancer cells with either SPFs or SMFs, were established to evaluate their active role on tumor progression and metastasis. RESULTS: We found that fibrosis with alpha smooth muscle actin (α-SMA) expression was a significant pathological feature of CMPI. The differences in proliferation and gene expression profile analyses suggested SPFs and SMFs were distinct populations, and that SPFs were characterized by a higher expressions of extracellular matrix (ECM)-associated genes. Furthermore, compared with SMFs, SPFs showed more variable alteration in gene expressions after cancer-cell-conditioned medium stimulation. Gene ontology analysis revealed that SPFs-specific upregulated genes were enriched by actin-binding or contractile-associated genes including α-SMA encoding ACTA2. Mouse xenograft tumors derived by co-injection of cancer cells with SPFs showed enhancement of tumor growth, metastasis, and capacity for tumor formation compared to those derived from co-injection with cancer cells and SMFs. CONCLUSIONS: CMPI is a special microenvironment, and interaction of SPFs and cancer cells within CMPI promote tumor growth and metastasis.