Platelet-derived growth factor (PDGF) cross-signaling via non-corresponding receptors indicates bypassed signaling in colorectal cancer.

Platelet-derived growth factor (PDGF) signaling, besides other growth factor-mediated signaling pathways like vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), seems to play a crucial role in tumor development and progression. We have recently provided evidence for upregulation of PDGF expression in UICC stage I-IV primary colorectal cancer (CRC) and demonstrated PDGF-mediated induction of PI3K/Akt/mTOR signaling in CRC cell lines. The present study sought to follow up on our previous findings and explore the alternative receptor cross-binding potential of PDGF in CRC. Our analysis of primary human colon tumor samples demonstrated upregulation of the PDGFRß, VEGFR1, and VEGFR2 genes in UICC stage I-III tumors. Immunohistological analysis revealed co-expression of PDGF and its putative cross-binding partners, VEGFR2 and EGFR. We then analyzed several CRC cell lines for PDGFRα, PDGFRß, VEGFR1, and VEGFR2 protein expression and found these receptors to be variably expressed amongst the investigated cell lines. Interestingly, whereas Caco-2 and SW480 cells showed expression of all analyzed receptors, HT29 cells expressed only VEGFR1 and VEGFR2. However, stimulation of HT29 cells with PDGF resulted in upregulation of VEGFR1 and VEGFR2 expression despite the absence of PDGFR expression and mimicked the effect of VEGF stimulation. Moreover, PDGF recovered HT29 cell proliferation under simultaneous treatment with a VEGFR or EGFR inhibitor. Our results provide some of the first evidence for PDGF cross-signaling through alternative receptors in colorectal cancer and support anti-PDGF therapy as a combination strategy alongside VEGF and EGF targeting even in tumors lacking PDGFR expression.

Expression of Tumor-mediated CD137 ligand in human colon cancer indicates dual signaling effects.

CD137-targeting immune therapy, which activates anti-tumor T effector cell responses, seems to be an attractive concept in clinical oncology. Recent evidence has demonstrated that tumor cells besides T cells and antigen-presenting cells are able to express CD137 and CD137L. Here we aimed to identify CD137/CD137L expression in established colon cancer cell lines and primary tumors (UICC stages I-IV) from patients with documented long-term follow-up. CD137/CD137L expression was highly upregulated in early to late-stage tumors while the inverse was observed in patient-derived peripheral blood mononuclear cells. High CD137L expression within primary tumors was mediated by tumor cells and significantly correlated with the occurrence of distant metastases and shortened survival in advanced stages of disease (UICC stage IV). Interestingly, induced tumor cell signaling via CD137L on its surface in vitro resulted in dual effects: (i) reduced tumor cell proliferation suggesting inhibitory signaling in all investigated cancers and (ii) increased epithelial-to-mesenchymal transition signaling events. Taken together CD137/CD137L expression was stage-dependently upregulated with shortened survival in patients with highly CD137L-expressing tumors. Our clinical and experimental data suggest that colon cancer cells predominantly express CD137L and thereby have negative impact on overall survival through a process of reverse signaling. Beside agonistic CD137 antibody therapy to foster T effector cell responses, CD137L-mediated intervention strategies may become instrumental to circumvent relapsed tumor growth through induced epithelial-to-mesenchymal transition and consecutive metastases formation.

Costimulation Blockade in Transplantation.

T cells play a pivotal role in orchestrating immune responses directed against a foreign (allogeneic) graft. For T cells to become fully activated, the T-cell receptor (TCR) must interact with the major histocompatibility complex (MHC) plus peptide complex on antigen-presenting cells (APCs), followed by a second "positive" costimulatory signal. In the absence of this second signal, T cells become anergic or undergo deletion. By blocking positive costimulatory signaling, T-cell allo-responses can be aborted, thus preventing graft rejection and promoting long-term allograft survival and possibly tolerance (Alegre ML, Najafian N, Curr Mol Med 6:843-857, 2006; Li XC, Rothstein DM, Sayegh MH, Immunol Rev 229:271-293, 2009). In addition, costimulatory molecules can provide negative "coinhibitory" signals that inhibit T-cell activation and terminate immune responses; strategies to promote these pathways can also lead to graft tolerance (Boenisch O, Sayegh MH, Najafian N, Curr Opin Organ Transplant 13:373-378, 2008). However, T-cell costimulation involves an incredibly complex array of interactions that may act simultaneously or at different times in the immune response and whose relative importance varies depending on the different T-cell subsets and activation status. In transplantation, the presence of foreign alloantigen incites not only destructive T effector cells but also protective regulatory T cells, the balance of which ultimately determines the fate of the allograft (Lechler RI, Garden OA, Turka LA, Nat Rev Immunol 3:147-158, 2003). Since the processes of alloantigen-specific rejection and regulation both require activation of T cells, costimulatory interactions may have opposing or synergistic roles depending on the cell being targeted. Such complexities present both challenges and opportunities in targeting T-cell costimulatory pathways for therapeutic purposes. In this chapter, we summarize our current knowledge of the various costimulatory pathways in transplantation and review the current state and challenges of harnessing these pathways to promote graft tolerance (summarized in Table 10.1).

ATP-binding cassette member B5 (ABCB5) promotes tumor cell invasiveness in human colorectal cancer.

ABC member B5 (ABCB5) mediates multidrug resistance (MDR) in diverse malignancies and confers clinically relevant 5-fluorouracil resistance to CD133-expressing cancer stem cells in human colorectal cancer (CRC). Because of its recently identified roles in normal stem cell maintenance, we hypothesized that ABCB5 might also serve MDR-independent functions in CRC. Here, in a prospective clinical study of 142 CRC patients, we found that ABCB5 mRNA transcripts previously reported not to be significantly expressed in healthy peripheral blood mononuclear cells are significantly enriched in patient peripheral blood specimens compared with non-CRC controls and correlate with CRC disease progression. In human-to-mouse CRC tumor xenotransplantation models that exhibited circulating tumor mRNA, we observed that cancer-specific ABCB5 knockdown significantly reduced detection of these transcripts, suggesting that the knockdown inhibited tumor invasiveness. Mechanistically, this effect was associated with inhibition of expression and downstream signaling of AXL receptor tyrosine kinase (AXL), a proinvasive molecule herein shown to be produced by ABCB5-positive CRC cells. Importantly, rescue of AXL expression in ABCB5-knockdown CRC tumor cells restored tumor-specific transcript detection in the peripheral blood of xenograft recipients, indicating that ABCB5 regulates CRC invasiveness, at least in part, by enhancing AXL signaling. Our results implicate ABCB5 as a critical determinant of CRC invasiveness and suggest that ABCB5 blockade might represent a strategy in CRC therapy, even independently of ABCB5's function as an MDR mediator.

Hypoxia induces differential expression patterns of osteopontin and CD44 in colorectal carcinoma.

The plasma protein osteopontin (OPN) is considered to be a tumor biomarker, where elevated plasma levels are associated with poor prognosis. Additionally, OPN is expressed in the presence of tumor hypoxia, which is an adverse prognostic factor in radiation oncology. One of its receptors, the proposed tumor stem cell marker CD44, is also associated with aggressive tumors, shown for example in colon cancer. The expression of CD44 and its splice variants (particularly CD44v6) can be upregulated by OPN itself. In the present study, we aimed to investigate the influence of hypoxia on the expression of OPN and its binding partners CD44 and CD44v6 in colon carcinoma cell lines in vitro, using SW480, SW620, HT29 and HCT116 cells. Additionally, we investigated the effect of irradiation on the expression pattern of OPN and its ligands, and the influence of hypoxia on the clonogenic survival of the cells after irradiation. While the expression patterns were nearly unaltered by irradiation, hypoxia led to an upregulation of OPN protein expression and an increase in the radioresistance in all tested colorectal carcinoma cell lines. However, a similar clear statement with regard to the expression of CD44 and CD44v6 is not possible. We hypothesize that the OPN receptors differ in their expression pattern between cell lines depending on the degree of their malignancy.

Clinical Significance of Disseminated Pluripotent Tumor Cell Signature Expression in the Bone Marrow from Patients with Colorectal Cancer.

PURPOSE: Disseminated tumor cells (DTCs) are critically involved in tumor relapse and survival in several invasive tumors. We previously showed that the ATP-binding cassette (ABC) transporter, ABCB5, is a chemoresistance mediator expressed on specific cell subsets in colorectal cancer (CRC) and other malignancies. This study evaluated the molecular signature expression and its clinical relevance of DTCs in bone marrow from patients with colon cancer. METHODS: This study included 49 consecutive patients (UICC stage I-IV) that underwent curatively intended or palliative surgery for CRC. We analyzed cells from bone marrow aspirates obtained before surgery and derived from patients that had completed minimally a 5-year follow-up. The gene expression of ABCB5 in comparison to CD133 (molecule for identifying cancer initiating cells), Lgr5 (an intestinal stem cell marker) as well as Cytokeratin (CK) 20 (terminally differentiated tumor cells of epithelial origin) in these cells was evaluated. RESULTS: Bone marrow analysis showed differential expression between the analyzed genes. ABCB5 and Lgr5 and to lesser extent CD133 and CK20 genes were significantly expressed in the analyzed cells from bone marrow aspirates while only ABCB5 and Lgr5 were significantly negative associated with tumor progress and overall survival. CONCLUSION: Overexpression of ABCB5 and Lgr5 in bone marrow negatively influenced patient survival pointing to a specific chemo resistant and pluripotent cell subgroup of DTCs in the bone marrow. ABCB5 like Lgr5 positive cells seem to be involved in limited tumor related patient survival, suggesting that ABCB5- and Lgr5-positive cells may be relevant for specific clinical intervention strategies.

Upregulated Heat Shock Proteins After Hyperthermic Chemotherapy Point to Induced Cell Survival Mechanisms in Affected Tumor Cells From Peritoneal Carcinomatosis.

In patients with peritoneal carcinomatosis cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) represents a promising treatment strategy. Here, we studied the role of hyperthermic chemotherapy on heat shock protein (HSP) expression and induction of tumor cell death and survival. HSP27, HSP70, and HSP90 combined with effects on tumor cell proliferation and chemosensitivity were analyzed in human colon cancer. Hyperthermic chemotherapy resulted in significant HSP27/HSP70 and HSP90 gene/protein overexpression in analyzed HT-29/SW480/SW620 colon cancer cells and peritoneal metastases from patients displaying amplified expression of proliferation markers, proliferating cell nuclear antigen and antiapoptotic protein Bcl-xL. Moreover, functionally increased chemoresistance against 5-fluorouracil/mitomycin C and oxaliplatin after hyperthermic chemotherapy points to induced survival mechanisms in cancer cells. In conclusion, the results indicate that intracellular HSP-associated antiapoptotic and proliferative effects after hyperthermic chemotherapy negatively influence beneficial effects of hyperthermic chemotherapy-induced cell death. Therefore, blocking HSPs could be a promising strategy to further improve the rate of tumor cell death and outcome of patients undergoing HIPEC therapy.

Toll Like Receptor 2, 4, and 9 Signaling Promotes Autoregulative Tumor Cell Growth and VEGF/PDGF Expression in Human Pancreatic Cancer.

Toll like receptor (TLR) signaling has been suggested to play an important role in the inflammatory microenvironment of solid tumors and through this inflammation-mediated tumor growth. Here, we studied the role of tumor cells in their process of self-maintaining TLR expression independent of inflammatory cells and cytokine milieu for autoregulative tumor growth signaling in pancreatic cancer. We analyzed the expression of TLR2, -4, and -9 in primary human cancers and their impact on tumor growth via induced activation in several established pancreatic cancers. TLR-stimulated pancreatic cancer cells were specifically investigated for activated signaling pathways of VEGF/PDGF and anti-apoptotic Bcl-xL expression as well as tumor cell growth. The primary pancreatic cancers and cell lines expressed TLR2, -4, and -9. TLR-specific stimulation resulted in activated MAP-kinase signaling, most likely via autoregulative stimulation of demonstrated TLR-induced VEGF and PDGF expression. Moreover, TLR activation prompted the expression of Bcl-xL and has been demonstrated for the first time to induce tumor cell proliferation in pancreatic cancer. These findings strongly suggest that pancreatic cancer cells use specific Toll like receptor signaling to promote tumor cell proliferation and emphasize the particular role of TLR2, -4, and -9 in this autoregulative process of tumor cell activation and proliferation in pancreatic cancer.

Exclusive inhibition of PI3K/Akt/mTOR signaling is not sufficient to prevent PDGF-mediated effects on glycolysis and proliferation in colorectal cancer.

Platelet-derived growth factor (PDGF) and signaling via its receptors plays a crucial role in tumor cell proliferation and thus may represent an attractive target besides VEGF/EGFR-based antibody therapies. In this study we analyzed the influence of PDGF in colorectal cancer. PDGF was expressed intensively in early and even more intensively in late stage primary CRCs. Like VEGF, PDGF enhanced human colon cancer proliferation, and increased oxidative glycolytic activity, and activated HIF1α and c-Myc in vitro. PDGF activated the PI3K/Akt/mTOR pathway while leaving MAPK signaling untouched. Further dissection showed that inhibition of Akt strongly impeded cancer cell growth while inhibition of PI3K did not. MAPK analysis suggested an inhibitory crosstalk between both pathways, thus explaining the different effects of the Akt and PI3K inhibitors on cancer cell proliferation. PDGF stimulates colon cancer cell proliferation, and prevents inhibitor induced apoptosis, resulting in tumor growth. Therefore inhibition of PDGF signaling seems to be a promising target in colorectal cancer therapy. However, due to the multifaceted nature of the intracellular PDGF signaling, careful intervention strategies are needed when looking into specific signaling pathways like PI3K/Akt/mTOR and MAPK.

TLR7 and TLR8 expression increases tumor cell proliferation and promotes chemoresistance in human pancreatic cancer.

Chronic inflammation as an important epigenetic and environmental factor for putative tumorigenesis and tumor progression may be associated with specific activation of Toll-like receptors (TLR). Recently, carcinogenesis has been suggested to be dependent on TLR7 signaling. In the present study, we determined the role of both TLR7 and TLR8 expression and signaling in tumor cell proliferation and chemoresistance in pancreatic cancer. Expression of TLR7/TLR8 in UICC stage I-IV pancreatic cancer, chronic pancreatitis, normal pancreatic tissue and human pancreatic (PANC1) cancer cell line was examined. For in vitro/in vivo studies TLR7/TLR8 overexpressing PANC1 cell lines were generated and analyzed for effects of (un-)stimulated TLR expression on tumor cell proliferation and chemoresistance. TLR expression was increased in pancreatic cancer, with stage-dependent upregulation in advanced tumors, compared to earlier stages and chronic pancreatitis. Stimulation of TLR7/TLR8 overexpressing PANC1 cells resulted in elevated NF-κB and COX-2 expression, increased cancer cell proliferation and reduced chemosensitivity. More importantly, TLR7/TLR8 expression increased tumor growth in vivo. Our data demonstrate a stage-dependent upregulation of both TLR7 and TLR8 expression in pancreatic cancer. Functional analysis in human pancreatic cancer cells point to a significant role of both TLRs in chronic inflammation-mediated TLR7/TLR8 signaling leading to tumor cell proliferation and chemoresistance.

The role of FOXP3 in disease progression in colorectal cancer patients.

The transcription factor forkhead box P3 (FOXP3) has been identified as a marker of CD4+CD25+ regulatory T cells and is a key determinant of their immunosuppressive functions. FOXP3 has indeed been shown to limit antitumor immune responses during tumor progression. In addition, by expressing FOXP3, tumor cells may evade effector T-cell responses.

Hyperthermic intraperitoneal chemotherapy in patients with peritoneal carcinomatosis: role of heat shock proteins and dissecting effects of hyperthermia.

BACKGROUND: In patients with isolated peritoneal carcinomatosis (PC) of gastrointestinal cancer, hyperthermic intraperitoneal chemotherapy (HIPEC) represents a promising treatment option integrated into multimodal concepts. Heat shock proteins (HSP) seem to play a major role in cellular stress during HIPEC therapy. We analyzed differentially hyperthermic conditions and HSPs responsible for cell stress-mediated repair mechanisms in tumor tissues from patients who underwent HIPEC therapy and in an in vitro hyperthermic model. METHODS: Tumor tissues from our patient cohort with isolated PC were selected for further analysis when representative material was available before and after HIPEC therapy. To further dissect the role of HSPs under conditions of hyperthermia, gene and protein expression was additionally determined, together with cellular apoptosis and proliferation in human HT-29 colon cancer cells. RESULTS: Differently up-regulated HSP70/72 and HSP90 gene and protein expression was found in all investigated patient tumors. In vitro studies confirmed observations from clinical tumor analysis as underlying HSP-mediated cell stress mechanisms. Moreover, results from proliferation and apoptosis assays combined with differentiated HSP expression analysis demonstrated the relevance of preselecting specific target temperatures to achieve optimal toxic effects on remaining tumor cells in vivo. CONCLUSIONS: Therapeutic approaches like HIPEC to achieve antiproliferative and apoptosis-inducing cellular effects in patients with PC are negatively influenced by highly conserved HSP mechanisms in tumor cells. This study shows for the first time that specific hyperthermic conditions are necessary to be established to achieve optimal toxic effects on tumor cells during HIPEC therapy, a finding that opens potentially new therapeutic strategies.

Expression of Foxp3 in colorectal cancer but not in Treg cells correlates with disease progression in patients with colorectal cancer.

BACKGROUND: Regulatory T cells (Treg) expressing the transcription factor forkhead-box protein P3 (Foxp3) have been identified to counteract anti-tumor immune responses during tumor progression. Besides, Foxp3 presentation by cancer cells itself may also allow them to evade from effector T-cell responses, resulting in a survival benefit of the tumor. For colorectal cancer (CRC) the clinical relevance of Foxp3 has not been evaluated in detail. Therefore the aim of this study was to study its impact in colorectal cancer (CRC). METHODS AND FINDINGS: Gene and protein analysis of tumor tissues from patients with CRC was performed to quantify the expression of Foxp3 in tumor infiltrating Treg and colon cancer cells. The results were correlated with clinicopathological parameters and patients overall survival. Serial morphological analysis demonstrated Foxp3 to be expressed in cancer cells. High Foxp3 expression of the cancer cells was associated with poor prognosis compared to patients with low Foxp3 expression. In contrast, low and high Foxp3 level in tumor infiltrating Treg cells demonstrated no significant differences in overall patient survival. CONCLUSIONS: Our findings strongly suggest that Foxp3 expression mediated by cancer cells rather than by Treg cells contribute to disease progression.