Current Molecular Medicine - Volume 13, Issue 3, 2013

Pancreatic neuroendocrine tumors (PNETs) are rare but are well understood to cover a broad spectrum of clinical presentation, tumor biology and prognosis. More than 60% of PNETs are diagnosed at advanced disease stage and are ineligible for surgical resection. Prior to 2011, streptozocin was the only approved agent for unresectable advanced PNETs. In recent years, breakthroughs in signal pathway research have led to the identification of new therapeutic targets and agents directed at the molecular level. In 2011, two new targeted therapeutic agents, sunitinib and everolimus, were approved by the Food and Drug Administration (FDA). Sunitinib is an inhibitor of multiple tyrosine kinases, and everolimus is an inhibitor of the mammalian target of rapamycin (mTOR) pathway. This review discusses the major signaling pathways that are frequently mutated or deregulated in PNETs, and the implications of molecular alterations for PNET therapy. Biologic therapy through targeting relevant pathways represents a promising approach in the therapy of advanced and unresectable PNETs.

CA 19-9 and CEA are the most commonly used biomarkers for diagnosis and management of patients with pancreatic cancer. Since the original compendium by Steinberg in 1990, numerous studies have reported the use of CA 19-9 and, to a lesser extent, CEA in the diagnosis of pancreatic cancer. Here we update an evaluation of the accuracy of CA 19-9 and CEA, and, unlike previous reviews, focus on discrimination between malignant and benign disease instead of normal controls. In 57 studies involving 3,285 pancreatic carcinoma cases, the combined sensitivity of CA 19-9 was 78.2% and in 37 studies involving 1,882 cases with benign pancreatic disease the specificity of CA 19-9 was 82.8%. From the combined analysis of studies reporting CEA, the sensitivity was 44.2% (1,324 cases) and the specificity was 84.8% (656 cases). These measurements more appropriately reflect the expected biomarker accuracy in the differential diagnosis of patients with periampullary diseases. We also present a summary of the use of CA 19-9 as a prognostic tool and evaluate CA 19-9 diagnostic and prognostic utility in a 10-year, single institution experience.

Glioblastoma (GBM) is a highly malignant primary brain tumor known for its invasiveness and aggressive resistance to standard treatment. It is currently the most common primary brain tumor which is associated with a high mortality rate. Tumor initiating cells (TICs) are a subpopulation of GBM stem cells which are capable of self-renewal and apoptotic resistance, and are thought to account for GBMs aggressive nature. Recent efforts have focused on therapies which target key intracellular apoptotic pathways which may confer tumor resistance, such as Akt, p53, Bcl-2 family proteins, caspase family proteases, and more recently microRNAs. Research into microRNA's role in GBM has shown that microRNAs play a key regulatory role in the GBM apoptotic pathway, making it a potential therapeutic target. In this review we summarized the molecular mechanisms involved in the signaling pathways of human GBM TIC apoptosis and microRNAs, the contemporary treatments involving different members of the signaling cascade, and the future direction of GBM treatment strategies.

The failure to control glioblastoma progression is a major challenge for neuro-oncologists. Emerging data indicate that genetic and epigenetic heterogeneities within tumor cells play a dominant role in the development of resistant disease. These heterogeneities develop because driver mutations enable the proliferation of certain clones of transformed cells within the tumor microenvironment while pre-existing passenger or secondary mutations emerge from the clonal selection process during treatment. In addition, epigenetic changes provide another means of modifying the existing heterogeneous genetic background of tumor cells. These cumulative changes create challenges for the detection, characterization and treatment of glioblastomas, but new opportunities allow the development of advanced diagnostic modalities and individualized therapies. Furthermore, mutations in the epidermal growth factor receptor (EGFR) alter binding capability to targeted agents like erlotinib, rendering it inactive to block EGFR signaling. Receptor class switching and tyrosine kinase decoupling from cell cycle machineries are also mechanisms that can render tumor cells resistant to EGFR blockade. Therefore, effective therapy most likely requires the combination of personalized medicine treatment offered by targeted drugs and less specific therapies that aim at other processes within the tumor microenvironment. The goal is to take advantage of the specificity offered by targeted drugs to block proliferation of tumor cells harboring driver mutations while less specific treatments can be used against cells with passenger mutations.

Tumor heterogeneity is recognized as a major issue within clinical oncology, and the concept of personalized molecular medicine is emerging as a means to mitigate this problem. Given the vast number of cancer types and subtypes, robust pre-clinical models of cancer must be studied to interrogate the molecular mechanisms involved in each scenario. In particular, mouse models of tumor metastasis are of critical importance for pre-clinical cancer research at the cancer cell molecular level. In many of these experimental systems, tumor cells are injected intravenously, and the distribution and proliferation of these cells are subsequently analyzed via ex vivo methods. These techniques require large numbers of animals coupled with time-consuming histological preparation and analysis. Herein, we demonstrate the use of two facile and noninvasive imaging techniques to enhance the study of a pre-clinical model of breast cancer metastasis in the lung. Breast cancer cells were labeled with a near-infrared fluorophore that enables their visualization. Upon injection into a living mouse, the distribution of the cells in the body was detected and measured using whole animal fluorescence imaging. X-ray computed tomography (CT) was subsequently used to provide a quantitative measure of longitudinal tumor cell accumulation in the lungs over six weeks. A nuclear probe for lung perfusion, 99mTc-MAA, was also imaged and tested during the time course using single photon emission computed tomography (SPECT). Our results demonstrate that optical fluorescence methods are useful to visualize cancer cell distribution patterns that occur immediately after injection. Longitudinal imaging with X-ray CT provides a convenient and quantitative avenue to measure tumor growth within the lung space over several weeks. Results with nuclear imaging did not show a correlation between lung perfusion (SPECT) and segmented lung volume (CT). Nevertheless, the combination of animal models and noninvasive optical and CT imaging methods provides better research tools to study cancer cell differences at the molecular level. Ultimately, the knowledge gleaned from these improved studies will aid researchers in uncovering the mechanisms mediating breast cancer metastasis, and eventually improve the treatments of patients in the clinic.

Pancreatic and duodenal homeobox-1 (PDX-1) is a homeodomain-containing transcription factor that plays a critical role in pancreatic development, β-cell differentiation, maintenance of normal β-cell function and tumorigenesis. PDX-1 is subjected to extensive post-translational modifications for its stability, subcellular location and transactivity. We report here that PDX-1 expression is up-regulated by p38 MAP kinase. Antibody array screen identified p38 as a candidate PDX-1-interacting protein in GFP-PDX-1 stable HEK293 cells. The p38-PDX-1 interaction was confirmed by immunoprecipitation/Western blotting analysis in both transient transfection system of HEK293 cells and endogenous system of β-TC-6 cells stimulated by glucagon-like peptide 1 (GLP-1). Co-transfection of p38 with PDX-1 resulted in increased PDX-1 expression in HEK293 cells, which was accompanied by a decreased PDX-1 ubiquitination. Mass spectrometry analysis showed that Ser 268 of human PDX-1 was phosphorylated in GFP-PDX-1 stable HEK293 cells. Functional mutagenesis analysis showed that mutation of Ser 269 of mouse PDX-1 (corresponding to Ser 268 of human PDX-1) into nonphosphorylatable alanine abolished the stabilizing effect of p38 on PDX-1, which was in line with enhanced PDX-1 ubiquitination and shortened half-life of PDX-1. p38 showed kinase activity towards PDX-1 in vitro, suggesting that Ser 269 is a potential p38-regulated phosphorylation site within PDX-1. GLP-1-stimulated PDX-1 expression was accompanied by p38 kinase activation in mouse insulinoma β-TC-6 cells and p38 inhibitor SB202190 inhibited GLP-1-stimulated PDX-1 expression with accompanied inhibition of p38 kinase activation. Taken together, our studies indicated that p38 MAP kinase is a positive regulator of PDX-1 stability and that p38 exerts its stabilizing effect on PDX-1 through a phosphorylation-dependent inhibition of PDX-1 ubiquitination.

Methyl-CpG binding domain protein 1 (MBD1) has been implicated in transcriptional regulation, heterochromatin formation, genomic stability, cell-cycle progression and development. It is also predicted that MBD1 might be involved in tumor development and progression. However, whether and how MBD1 is involved in tumorigenesis, especially in pancreatic cancer (PC), is currently unknown. We found that MBD1 was significantly up-regulated in PC tissues compared with the surrounding normal tissues according to RT-PCR data. Tissue microarray (TMA) based immunohistochemical study from 58 surgically resected PC specimens indicated that higher MBD1 expression correlated with lymph node metastasis and poor survival in PC patients. Gain- and loss-of-function studies in vitro validated MBD1 as a potent oncogene promoting PC cell invasion as well as epithelial-mesenchymal transition (EMT). Mechanistically, MBD1 is associated with Twist and NAD-dependent deacetylase sirtuin-1 (SIRT1), thereby forming the Twist-MBD1-SIRT1 complex on the CDH1 promoter, which resulted in reduced E-cadherin transcription activity and increased cell EMT ability. Significantly, targeting MBD1 reversed the EMT phenotype of PC and restored sensitivity to chemotherapy. Taken together, the results of our study revealed a novel function of MBD1 in PC invasion and metastasis by providing a molecular mechanism underlying MBD1-promoted EMT. Thus MBD1 may serve as a potential therapeutic target for PC.

Deregulated expression of zinc transporters was linked to several cancers. However, the detailed expression profile of all human zinc transporters in normal human organs and in human cancer, especially in pancreatic cancer is not available. The objectives of this study are to investigate the complete expression patterns of 14 ZIP and 10 ZnT transporters in a large number of normal human organs and in human pancreatic cancer tissues and cell lines. We examined the expression patterns of ZIP and ZnT transporters in 22 different human organs and tissues, 11 pairs of clinical human pancreatic cancer specimens and surrounding normal/benign tissues, as well as 10 established human pancreatic cancer cell lines plus normal human pancreatic ductal epithelium (HPDE) cells, using real time RT-PCR and immunohistochemistry. The results indicate that human zinc transporters have tissue specific expression patterns, and may play different roles in different organs or tissues. Almost all the ZIPs except for ZIP4, and most ZnTs were down-regulated in human pancreatic cancer tissues compared to the surrounding benign tissues. The expression patterns of individual ZIPs and ZnTs are similar among different pancreatic cancer lines. Those results and our previous studies suggest that ZIP4 is the only zinc transporter that is significantly up-regulated in human pancreatic cancer and might be the major zinc transporter that plays an important role in pancreatic cancer growth. ZIP4 might serve as a novel molecular target for pancreatic cancer diagnosis and therapy.

CXC chemokine receptor type 4 (CXCR4) plays a prominent role in cancer progression and metastasis. However, its association with breast cancer subtypes is still unknown. In the current study, we analyzed the expression level and the cellular location of CXCR4 in 175 cases of human breast tumors, including 75 cases of triple-negative breast cancers (TNBCs), 41 cases of luminal-subtypes and 60 cases of HER2-positive breast cancers by using immmunohistochemistry (IHC). We found that CXCR4 was expressed more frequently in the TNBCs than in other subtypes (71% for TNBC vs 44% for HER2-positive and 37% for luminal subtype, p<0.001). In the TNBC group, CXCR4 positive patients have a significantly higher rate of visceral metastasis (liver, lung and brain). The expression level of CXCR4 is also significantly related to tumor size, advanced TNM stage, shorter overall- and disease-free survival. However, in luminal or HER2-positive breast cancer groups, CXCR4 is not correlated with such clinico-pathological characteristics and survival. Taken together, our data indicate that CXCR4 might exert its function exclusively in TNBC patients, suggesting that targeting CXCR4 might be an effective therapy for TNBC patients.

The correlation between the loss of Profilin 1 (Pfn1) with tumor progression indicated that Pfn1 is a tumor suppressor in human carcinoma. The molecular mechanisms underlying Pfn1 tumor suppression has yet to be elucidated. In this study, we showed that Pfn1 overexpression sensitizes cancer cells to apoptosis through the typical intrinsic apoptotic pathway. Mechanistically, the increased Pfn1 expression mediated the upregulation of p53R273H, one of the most common tumor-associated hotspot mutations of p53, with transactivation deletion in tumorigenesis and increased localization of p53R273H in cytoplasm. Further studies showed that mutant p53R273H was involved in apoptosis induced by Staurosporine (STS) via transcriptionindependent mitochondrial functions. We observed (i) the increased cytosolic localization of p53R273H, (ii) the activation of phosphorylation at Ser15, (iii) its mitochondrial localization; Pfn1 acted as a positive regulator of these processes. We also found that Pfn1 interacted with p53R273H and thus facilitated its exertion over the transcription-independent activity in the cytoplasm during drug action. Our results define a new function and mechanism of Pfn1 demonstrating that the combined effect with apoptotic agents led to a synergistic increase in apoptosis. In addition, p53R273H abrogating DNA binding was found to play a major role in the Pfn1- sensitized apoptosis through a transactivation-independent and cytosolic activity.

Recently immunoglobulin G (IgG) was found to be produced by neoplasms and promote tumor growth in cancer cell lines and animal models. To investigate the pathophysiological significance of cancerproduced IgG in breast cancer, we examined the expressions of IgG in 68 breast cancers including 40 primary cancers without metastasis and 28 cancers with axillary lymph node metastases. IgG gene expression was detected in all these samples. We found that IgG-expressing cancer cells were predominantly located in the periphery of the primary cancer nest and that these cells showed more cellular atypia and nuclear pleomorphism. We also found that the abundance of IgG-expressing cancer cells was higher and the cells were more evenly distributed in the metastatic cancer cells than that in the primary lesion. These findings suggest that IgG-expressing breast cancer cells have a more aggressive biological behavior than the IgG negative cancer cells and it could be an indicator for progression and metastasis of the disease. Colocalization of IgG and C1q complement was detected in both primary and metastatic lesions implying that immune complexes might be formed in situ. We speculate that such immune complexes might facilitate immune escape of cancer cells. Our findings suggest that locally produced IgG plays important roles in breast cancer, and may serve as a potential therapeutic target.

The human Distal-less Homeobox (DLX) gene family encodes homeobox transcription factors involved in the control of morphogenesis and tissue homeostasis, which is primarily expressed in embryonic development. Recently, DLX gene family was reported to have essential roles in carcinogenesis. We have profiled whole genome expressed genes in 83 glioblastoma multiforme (GBM) patients from the Chinese Glioma Genome Atlas (CGGA) Group. Two major groups of samples were identified in mRNA expression profiles (referred to as Cluster 1 (C1) and Cluster 2 (C2)). We identified 7 out of the top 10 Gene Ontology terms in the C1 group were associated with differentiation and development of neuronal cell. The most significant prognostic gene was DLX2 (P<0.001, OR=1.744); overexpression of DLX2 indicated poor survival in the 83 GBM patients (low DLX2 vs high DLX2, 77.6 vs 44.7 weeks, P<0.001). Annotation of mRNA profiling data on GBM from The Cancer Genome Atlas and MD Anderson Cancer Center showed the proneural and neural subtypes highly correlated with low and high DLX2 expression, respectively. Knocking down of DLX2 in GBM cell line-LN229 results in decreased cyclin D1 expression and cell proliferation. Collectively, these data identified high expression of DLX2 as a poor prognostic marker to GBM patients.

Breast cancer is a heterogenetic tumor at the cellular level with multiple factors and components. The inconsistent expression of molecular markers during disease progression reduces the accuracy of diagnosis and efficacy of target-specific therapy. Single target-specific imaging agents can only provide limited tumor information at one time point. In contrast, multiple target-specific imaging agents can increase the accuracy of diagnosis. The aim of this study was to demonstrate the ability of multi-agent imaging to discriminate such differences in single tumor. Mice bearing human cancer cell xenografts were tested to determine individual differences under optimal experimental conditions. Neovasculature agent (RGD peptide), tumor stromal agent (matrix metalloproteinase), and tumor cell markers (epidermal growth factor, Her-2, interleukin 11) imaging agents were labeled with reporters. 18F-Fluorodeoxyglucose was used to evaluate the tumor glucose status. Optical, X-ray, positron emission tomography, and computer tomography imaging modalities were used to determine tumor characteristics. Tumor size and imaging data demonstrated that individual differences exist under optimal experimental conditions. The target-specific agents used in the study bind to human breast cancer cell lines in vitro and xenografts in vivo. The pattern of binding corresponds to that of tumor markers. Multi-agent imaging had complementary effects in tumor detection. Multiple noninvasive imaging agents and modalities are complementary in the interrogation of unique biological information from each individual tumor. Such multi-agent approaches provide methods to study several disease components simultaneously. In addition, the imaging results provide information on disease status at the molecular level.

Breast cancer stem cells (BCSC) exist within many types of breast cancers, functioning to initiate tumorigenesis and augment its progression. The protein profile associated with BCSC has yet to be extensively studied. Mammospheres have been widely employed as a model system to study BCSC. We used proteomics on the MCF-7 breast cancer cell line to compare protein expression in mammosphere-derived cells to that of parental monolayer cells. We identified 34 differentially expressed proteins, seven of which were overexpressed, with the remaining downregulated in mammosphere-derived cells. These differentially expressed proteins include those involved in cell metabolism such as GAPDH and fatty acid synthase, stress response proteins like Hsp27 and FKBP4, and signal transduction related proteins like GIPC1. The expression of breast cancer tumorigenesis and progression-promoting proteins GAPDH and FKBP4 were validated through western blotting. These two proteins are especially recognized for their role in breast cancer resistance to current chemotherapies. The data generated by mammosphere proteomics suggest that this system can identify novel targets for breast cancer stem cells and may provide insights into novel therapy of breast cancer.

A proliferation-inducing ligand (APRIL) is overexpressed in many cancers such as colorectal, gastric and liver. Silence APRIL gene or neutralize its expression may serve as therapeutic strategies to manage those cancers. Current phase I clinical trial shows that heavily pretreated patients were well tolerated high dose intravenous infusion. Herein, we report a humanized antibody (Ab) that neutralizes APRIL protein and inhibits cancer cell proliferation both in vitro and in vivo. The Ab was generated through conjugating foreign immunodominant T-helper cell (Th) epitopes to the N- or C-terminal of four soluble APRIL mutants. In vitro anti-proliferation effects were evaluated in both Raji and Jurkat cell lines. In vivo inhibition of tumor growth was determined in a tumor xenograft animal model with green fluorescent protein (GFP) positive Raji cell line. In vitro data demonstrated that this Ab significantly inhibited both Raji and Jurkat cell proliferation in a dosedependent manner. The molecular imaging data demonstrated in vivo tumor growth delay when animals were treated with Ab. In conclusion, we have developed a humanized anti-APRIL Ab, which is effective in inhibiting tumor cell proliferations both in vitro and vivo. The humanized Ab has potential to reduce the immunoresponse in the clinical situation. This Ab also can be used in other APRIL mediated diseases such as Sjogren syndrome, multiple sclerosis and systemic lupus erythematosus.