Characterization of the mechanisms underlying the crosstalk between galectins and notch in gastric cancer

Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related deaths worldwide. Galectins form a family of β-galactosides binding proteins that recognize a variety of glycan-containing proteins at the cell surface and are overexpressed in various tumors, including gastric cancer. Galectins overexpression as well as changes in their subcellular distribution has been associated with gastric cancer progression and poor prognosis. It is not well understood, however, how the interaction between galectins and glycosylated receptors modulates tumor development and growth. Since Notch receptors and ligands contain glycan structures known to bind galectins, we aim to demonstrate that galectins expression in the tumor microenvironment may interfere with Notch signaling activation during tumor development and progression. Materials and methods Immunoprecipitation procedures with gastric cancer cell line AGS (ATCC CRL-1739) and MKN45 (ACC 409) were used to test for association between galectin-1/-3 and Notch-1 receptor. Furthermore, we transfected AGS cell line with siRNA against galectin-1/-3 or scramble using standard protocols (IDT DNA technologies), stimulate them with immobilized human recombinant delta-4 or Jagged-1 and assessed Notch-1 receptor activation. Results Galectin-1 and -3 interact with Notch-1 receptor and differentially modulate Notch signaling pathway upon activation by Delta/Jagged ligands. Galectin-1 knockdown alters Notch-1 activation induced by Delta-4 whereas galectin-3 knockdown alters jagged-1-mediated Notch-1 activation. Furthermore, we found that exogenously added galectin-3 can enhance Notch-1 activation by Jagged-1. Conclusion Our results suggest that galectin-1 and -3 interact with Notch-1 receptor and differentially modulate Notch signaling activation induced by Jagged-1 and Delta-4.

CCL20/CCR6 expression profile in pancreatic cancer

CCL20 and its receptor CCR6 have been shown to play a role in the onset, development and metastatic spread of various gastrointestinal malignancies. In this study, the expression profile and clinical significance of the CCL20/CCR6 system in distinct benign, pre-malignant and malignant pancreatic tissues was investigated.

FOXA1 Promotes Tumor Progression in Prostate Cancer and Represents a Novel Hallmark of Castration-Resistant Prostate Cancer

Forkhead box protein A1 (FOXA1) modulates the transactivation of steroid hormone receptors and thus may influence tumor growth and hormone responsiveness in prostate cancer. We therefore investigated the correlation of FOXA1 expression with clinical parameters, prostate-specific antigen (PSA) relapse-free survival, and hormone receptor expression in a large cohort of prostate cancer patients at different disease stages. FOXA1 expression did not differ significantly between benign glands from the peripheral zone and primary peripheral zone prostate carcinomas. However, FOXA1 was overexpressed in metastases and particularly in castration-resistant cases, but was expressed at lower levels in both normal and neoplastic transitional zone tissues. FOXA1 levels correlated with higher pT stages and Gleason scores, as well as with androgen (AR) and estrogen receptor expression. Moreover, FOXA1 overexpression was associated with faster biochemical disease progression, which was pronounced in patients with low AR levels. Finally, siRNA-based knockdown of FOXA1 induced decreased cell proliferation and migration. Moreover, in vitro tumorigenicity was inducible by ARs only in the presence of FOXA1, substantiating a functional cooperation between FOXA1 and AR. In conclusion, FOXA1 expression is associated with tumor progression, dedifferentiation of prostate cancer cells, and poorer prognosis, as well as with cellular proliferation and migration and with AR signaling. These findings suggest FOXA1 overexpression as a novel mechanism inducing castration resistance in prostate cancer.

Targeting phosphoinositide 3-kinase signalling in lung cancer

Lung cancer is the leading cause of cancer-related mortality worldwide and more than 1 million people annually die in consequence of lung cancer. Although an improvement in lung cancer treatment could be achieved, especially in the last decade, the development of additional therapeutic strategies is urgently required in order to provide improved survival benefit for patients. Lung cancer formation is caused by genetic modifications commonly caused by tobacco smoking. Numerous studies have demonstrated the role of extracellular growth factors in lung cancer cell proliferation, metastasis, and chemoresistance. Mutations and amplifications in molecules related to receptor tyrosine signalling, such as EGFR, ErbB2, c-Met, c-Kit, VEGFR, PI3K, and PTEN are only some of the alterations known to contribute to the development of lung cancer. The phosphoinositide 3-kinase (PI3K) pathway, fundamental for cell development, growth, and survival, is known to be frequently altered in neoplasia, including carcinomas of the lung. Based on the high frequency of alterations, which include mutations and amplifications, leading to over-activation of certain upstream/downstream mediators, targeting components of the PI3K signalling pathway is considered to be a promising therapeutic approach in cancer treatment. In this article we will summarize the current knowledge about the involvement of PI3K signalling in lung cancer and discuss the development of targeted therapies involving PI3K pathway inhibitors.

High NRBP1 expression in prostate cancer is linked with poor clinical outcomes and increased cancer cell growth

We recently established the rationale that NRBP1 (nuclear receptor binding protein 1) has a potential growth-promoting role in cell biology. NRBP1 interacts directly with TSC-22, a potential tumor suppressor gene that is differently expressed in prostate cancer. Consequently, we analyzed the role of NRBP1 expression in prostate cancer cell lines and its expression on prostate cancer tissue microarrays (TMA).

[Bone metastasis in prostate cancer]

Bone metastasis and skeletal complications have a devastating impact on the quality of life and are a major cause of morbidity in prostate cancer patients. In addition to established bone-targeted therapies, new drugs such as endothelin A receptor antagonists, MET and VEGFR-2 antagonists or radiopharmaceuticals are in the focus of development. The standard care in prostate cancer patients with bone metastases to prevent skeletal-related events (SRE) are bisphosphonates. Denosumab, a human monoclonal antibody against RANKL, appeared to be superior to zoledronic acid for prevention of SRE and has been shown to prolong bone metastases-free survival. In contrast to zoledronic acid, denosumab clearance is not dependent on kidney function and can be administered subcutaneously. Similar rates of toxicity were observed for both substances; however, long-term data for denosumab are limited.

Inhibition of the AKT/mTOR and erbB pathways by gefitinib, perifosine and analogs of gonadotropin-releasing hormone I and II to overcome tamoxifen resistance in breast cancer cells

Endocrine resistance in breast cancer remains a major clinical problem and is caused by crosstalk mechanisms of growth factor receptor cascades, such as the erbB and PI3K/AKT pathways. The possibilities a single breast cancer cell has to achieve resistance are manifold. We developed a model of 4-hydroxy-tamoxifen (OHT)‑resistant human breast cancer cell lines and compared their different expression patterns, activation of growth factor receptor pathways and compared cells by genomic hybridization (CGH). We also tested a panel of selective inhibitors of the erbB and AKT/mTOR pathways to overcome OHT resistance. OHT‑resistant MCF-7-TR and T47D-TR cells showed increased expression of HER2 and activation of AKT. T47D-TR cells showed EGFR expression and activated MAPK (ERK-1/2), whereas in resistant MCF-7-TR cells activated AKT was due to loss of CTMP expression. CGH analyses revealed remarkable aberrations in resistant sublines, which were predominantly depletions. Gefitinib inhibited erbB signalling and restored OHT sensitivity in T47D-TR cells. The AKT inhibitor perifosine restored OHT sensitivity in MCF-7-TR cells. All cell lines showed expression of receptors for gonadotropin-releasing hormone (GnRH) I and II, and analogs of GnRH-I/II restored OHT sensitivity in both resistant cell lines by inhibition of erbB and AKT signalling. In conclusion, mechanisms to escape endocrine treatment in breast cancer share similarities in expression profiling but are based on substantially different genetic aberrations. Evaluation of activated mediators of growth factor receptor cascades is helpful to predict response to specific inhibitors. Expression of GnRH-I/II receptors provides multi-targeting treatment strategies.

Molecular characterization of the porcine deleted in malignant brain tumors 1 gene (DMBT1)

The human gene deleted in malignant brain tumors 1 (DMBT1) is considered to play a role in tumorigenesis and pathogen defense. It encodes a protein with multiple scavenger receptor cysteine-rich (SRCR) domains, which are involved in recognition and binding of a broad spectrum of bacterial pathogens. The SRCR domains are encoded by highly homologous repetitive exons, whose number in humans may vary from 8 to 13 due to genetic polymorphism. Here, we characterized the porcine DMBT1 gene on the mRNA and genomic level. We assembled a 4.5 kb porcine DMBT1 cDNA sequence from RT-PCR amplified seminal vesicle RNA. The porcine DMBT1 cDNA contains an open reading frame of 4050 nt. The transcript gives rise to a putative polypeptide of 1349 amino acids with a calculated mass of 147.9 kDa. Compared to human DMBT1, it contains only four N-terminal SRCR domains. Northern blotting revealed transcripts of approximately 4.7 kb in size in the tissues analyzed. Analysis of ESTs suggested the existence of secreted and transmembrane variants. The porcine DMBT1 gene spans about 54 kb on chromosome 14q28-q29. In contrast to the characterized cDNA, the genomic BAC clone only contained 3 exons coding for N-terminal SRCR domains. In different mammalian DMBT1 orthologs large interspecific differences in the number of SRCR exons and utilization of the transmembrane exon exist. Our data suggest that the porcine DMBT1 gene may share with the human DMBT1 gene additional intraspecific variations in the number of SRCR-coding exons.

[Molecular targets in colon cancer]

Colorectal cancer is the second leading cause of cancer death in Switzerland. The nihilism that dominated the treatment of these patients for decades has been replaced by a measure of enthusiasm, given recent therapeutic advances. New anticancer drugs such as irinotecan and oxaliplatin have changed the standard chemotherapy treatment of metastatic colorectal cancer. However, the real hype has come from molecular targeted therapy. Identification of cellular processes characteristic of colon cancer has permitted therapeutic targeting with favorable therapeutic index. Inhibition of the epidermal growth factor receptor in the clinic has provided proof of principle that interruption of signal transduction cascades in patients has therapeutic potential. Angiogenesis, especially the vascular endothelial growth factor pathway, has been proven to be another highly successful molecular target. In this article, we will review molecular targets, which are under active clinical investigation in colon cancer.

NPY receptors in human cancer: a review of current knowledge

Many peptide hormone receptors are over-expressed in human cancer, permitting an in vivo targeting of tumors for diagnostic and therapeutic purposes. NPY receptors are novel and promising candidates in this field. Using in vitro receptor autoradiography, Y1 and Y2 receptors have been found to be expressed in breast carcinomas, adrenal gland and related tumors, renal cell carcinomas, and ovarian cancers in both tumor cells and tumor-associated blood vessels. Pathophysiologically, tumoral NPY receptors may be activated by endogenous NPY released from intratumoral nerve fibers or tumor cells themselves, and mediate NPY effects on tumor cell proliferation and tumoral blood supply. Clinically, tumoral NPY receptors may be targeted with NPY analogs coupled with adequate radionuclides or cytotoxic agents for a scintigraphic tumor imaging and/or tumor therapy.

Wild-type and splice-variant secretin receptors in lung cancer: overexpression in carcinoid tumors and peritumoral lung tissue

Gastrointestinal peptide hormone receptors, like somatostatin receptors, are often overexpressed in human cancer, allowing receptor-targeted tumor imaging and therapy. A novel candidate for these applications is the secretin receptor recently identified in pancreatic and cholangiocellular carcinomas. In the present study, secretin receptors were assessed in a non-gastrointestinal tissue, the human lung. Non-small-cell lung cancers (n=26), small-cell lung cancers (n=10), bronchopulmonary carcinoid tumors (n=29), and non-neoplastic lung (n=46) were investigated for secretin receptor protein expression with in vitro receptor autoradiography, using (125)I-[Tyr(10)] rat secretin and for secretin receptor transcripts with RT-PCR. Secretin receptor protein expression was found in 62% of bronchopulmonary carcinoids in moderate to high density, in 12% of non-small cell lung cancers in low density, but not in small cell lung cancers. In tumors found to be secretin receptor positive by autoradiography, RT-PCR revealed transcripts for the wild-type secretin receptor and for novel secretin receptor splice variants. In the non-neoplastic lung, secretin receptor protein expression was observed in low density along the alveolar septa in direct tumor vicinity in cases of acute inflammation, but not in histologically normal lung. In the autoradiographically positive peritumoral lung, RT-PCR showed transcripts for the wild-type secretin receptor and for a secretin receptor spliceoform different from those occurring in lung and gut tumors. In conclusion, secretin receptors are new markers for bronchopulmonary carcinoid tumors, and represent the molecular basis for an in vivo targeting of carcinoid tumors for diagnosis and therapy. Furthermore, secretin receptors may play a role in peritumoral lung pathophysiology. Secretin receptor mis-splicing specifically occurs in tumor and non-tumor lung pathology.

Simultaneous quantitative detection of relevant biomarkers in breast cancer by quantitative real-time PCR

The assessment of ERa, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERa, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERa, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Clinical impact of programmed cell death ligand 1 expression in colorectal cancer

BACKGROUND Programmed cell death 1 (PD-1) receptor triggering by PD ligand 1 (PD-L1) inhibits T cell activation. PD-L1 expression was detected in different malignancies and associated with poor prognosis. Therapeutic antibodies inhibiting PD-1/PD-L1 interaction have been developed. MATERIALS AND METHODS A tissue microarray (n=1491) including healthy colon mucosa and clinically annotated colorectal cancer (CRC) specimens was stained with two PD-L1 specific antibody preparations. Surgically excised CRC specimens were enzymatically digested and analysed for cluster of differentiation 8 (CD8) and PD-1 expression. RESULTS Strong PD-L1 expression was observed in 37% of mismatch repair (MMR)-proficient and in 29% of MMR-deficient CRC. In MMR-proficient CRC strong PD-L1 expression correlated with infiltration by CD8(+) lymphocytes (P=0.0001) which did not express PD-1. In univariate analysis, strong PD-L1 expression in MMR-proficient CRC was significantly associated with early T stage, absence of lymph node metastases, lower tumour grade, absence of vascular invasion and significantly improved survival in training (P=0.0001) and validation (P=0.03) sets. A similar trend (P=0.052) was also detectable in multivariate analysis including age, sex, T stage, N stage, tumour grade, vascular invasion, invasive margin and MMR status. Interestingly, programmed death receptor ligand 1 (PDL-1) and interferon (IFN)-γ gene expression, as detected by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in fresh frozen CRC specimens (n=42) were found to be significantly associated (r=0.33, P=0.03). CONCLUSION PD-L1 expression is paradoxically associated with improved survival in MMR-proficient CRC.

Multidrug resistance protein 4 (MRP4) expression in prostate cancer is associated with androgen signaling and decreases with tumor progression

Multidrug resistance protein 4 (MRP4) is a transmembrane transport protein found in many cell types and is involved in substrate-specific transport of endogenous and exogenous substrates. Recently, it has shown to be expressed in prostate cancer cell lines and to be among the most commonly upregulated transcripts in prostate cancer, although a comprehensive expression analysis is lacking so far. We aimed to investigate its expression by immunohistochemistry in a larger cohort of neoplastic and nonneoplastic prostate tissues (n = 441) and to correlate its expression with clinicopathological parameters including PSA-free survival times and molecular correlates of androgen signaling (androgen receptor (AR), prostate-specific antigen (PSA), and forkhead box A (FoxA)). MRP4 is widely expressed in benign and neoplastic prostate epithelia, but its expression gradually decreases during tumor progression towards castrate-resistant disease. Concordantly, it correlated with conventional prognosticators of disease progression and-within the group of androgen-dependent tumors-with AR and FoxA expression. Moreover, lower levels of MRP4 expression were associated with shorter PSA relapse-free survival times in the androgen-dependent group. In benign tissues, we found zone-dependent differences of MRP4 expression, with the highest levels in the peripheral and central zones. Although MRP4 is known to be regulated in prostate cancer, this study is the first to demonstrate a gradual downregulation of MRP4 protein during malignant tumor progression and a prognostic value of this loss of expression.

Investigation of IL23 (p19, p40) and IL23R highlights nuclear IL23p19 expression as a marker of indolent tumor features and favorable outcome in colorectal cancer patients

Objective: IL23 is involved in chronic inflammation but its role in cancer progression is not fully elucidated. Here we characterize IL23 subunits p40, p19 and IL23 receptor (IL23R) in the normal-adenoma-carcinomametastasis cascade of colorectal cancers and their relationship to clinicopathological and outcome data. Method: Immunohistochemistry for IL23R, IL12p40, IL23 and IL23p19 (monoclonal) was performed on a multi-punch tissue microarray (n=213 patients). Expression differences between normal-adenomas-cancerslymph nodes were evaluated. Correlation with clinicopathological and outcome data was undertaken. Results were validated on an independent cohort (n=341 patients). Results: An increased expression from normal-adenoma-cancer was observed (p<0.0001; all) followed by a marked reduction in lymph nodes (p<0.0001; all). Cytoplasmic and/or membranous staining of all markers was unrelated to outcome. Nuclear IL23p19 staining occurred in 23.1%and was associated with smaller tumor diameter (p=0.0333), early pT (p=0.0213), early TNM (p=0.0186), absence of vascular (p=0.0124) and lymphatic invasion (p=0.01493) and favorable survival (univariate (p=0.014) and multivariable (p=0.0321) analysis). All IL23p19 positive patients were free of distant metastasis (p=0.0146). Survival and metastasis results could be validated in Cohort 2. Conclusion: The presence of nuclear IL23p19 is related to indolent tumor features and favorable outcome supporting a more ‘protective’ role of this protein in colorectal cancer progression