Sukhwinder Kaur

Tumour exosome integrins determine organotropic metastasis.

Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

The multifaceted roles of neutrophil gelatinase associated lipocalin (NGAL) in inflammation and cancer.

Neutrophil gelatinase associated lipocalin (NGAL), also known as oncogene 24p3, uterocalin, siderocalin or lipocalin 2, is a 24kDa secreted glycoprotein originally purified from a culture of mouse kidney cells infected with simian virus 40 (SV-40). Subsequent investigations have revealed that it is a member of the lipocalin family of proteins that transport small, hydrophobic ligands. Since then, NGAL expression has been reported in several normal tissues where it serves to provide protection against bacterial infection and modulate oxidative stress. Its expression is also dysregulated in several benign and malignant diseases. Its small size, secreted nature and relative stability have led to it being investigated as a diagnostic and prognostic biomarker in numerous diseases including inflammation and cancer. Functional studies, conducted primarily on lipocalin 2 (Lcn2), the mouse homologue of human NGAL have revealed that Lcn2 has a strong affinity for iron complexed to both bacterial siderophores (iron-binding proteins) and certain human proteins like norepinephrine. By sequestering iron-laden siderophores, Lcn2 deprives bacteria of a vital nutrient and thus inhibits their growth (bacteriostatic effect). In malignant cells, its proposed functions range from inhibiting apoptosis (in thyroid cancer cells), invasion and angiogenesis (in pancreatic cancer) to increasing proliferation and metastasis (in breast and colon cancer). Ectopic expression of Lcn2 also promotes BCR-ABL induced chronic myelogenous leukemia in murine models. By transporting iron into and out of the cell, NGAL also regulates iron responsive genes. Further, it stabilizes the proteolytic enzyme matrix metalloprotease-9 (MMP-9) by forming a complex with it, and thereby prevents its autodegradation. The factors regulating NGAL expression are numerous and range from pro-inflammatory cytokines like interleukins, tumor necrosis factor-α and interferons to vitamins like retinoic acid. The purpose of this review article is to examine the expression, structure, regulation and biological role of NGAL and critically assess its potential as a novel diagnostic and prognostic marker in both benign and malignant human diseases.

Membrane-bound mucins: The mechanistic basis for alterations in the growth and survival of cancer cells

Mucins (MUC) are high molecular weight O-linked glycoproteins whose primary functions are to hydrate, protect, and lubricate the epithelial luminal surfaces of the ducts within the human body. The MUC family is comprised of large secreted gel forming and transmembrane (TM) mucins. MUC1, MUC4, and MUC16 are the well-characterized TM mucins and have been shown to be aberrantly overexpressed in various malignancies including cystic fibrosis, asthma, and cancer. Recent studies have uncovered the unique roles of these mucins in the pathogenesis of cancer. These mucins possess specific domains that can make complex associations with various signaling pathways, impacting cell survival through alterations of cell growth, proliferation, death, and autophagy. The cytoplasmic domain of MUC1 serves as a scaffold for interaction with various signaling proteins. On the other hand, MUC4 mediates its effect by stabilizing and enhancing the activity of growth factor receptor ErbB2. MUC16, previously known as CA125, is a well-known serum marker for the diagnosis of ovarian cancer and has a key role in stimulation and dissemination of ovarian cancer cells by interacting with mesothelin and galectin. Therefore, herein we discuss the function and divergent mechanisms of MUC1, MUC4, and MUC16 in carcinogenesis in the context of alteration in cell growth and survival.

Detection of the Potential Pancreatic Cancer Marker MUC4 in Serum Using Surface-Enhanced Raman Scattering

Pancreatic cancer (PC) is one of the most lethal malignancies. It has a 5-year survival rate of only 6%, owing in part to the lack of a reliable tumor marker for early diagnosis. Recent research has shown that the mucin protein MUC4 is aberrantly expressed in pancreatic adenocarcinoma cell lines and tissues but is undetectable in normal pancreas and chronic pancreatitis. Thus, the level of MUC4 in patient sera has the potential to function as a diagnostic and prognostic marker for PC. However, the measurement of MUC4 in sera using conventional test platforms (e.g., enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA)) has been unsuccessful. This has prevented the assessment of the utility of this protein as a possible PC marker in sera. In addressing this obstacle, the work herein examines the potential to create a simple diagnostic test for MUC4 through the development of a surface-enhanced Raman scattering (SERS)-based immunoassay, which was then used to demonstrate the first ever detection of MUC4 in cancer patient serum samples. Importantly, these measurements showed that sera from patients with PC produced a significantly higher SERS response for MUC4 compared to sera from healthy individuals and from patients with benign diseases. These results indicate that a SERS-based immunoassay can monitor MUC4 levels in patient sera, representing a much needed first step toward assessing the potential of this protein to serve as a serum marker for the early stage diagnosis of PC. This paper details these and other findings (i.e., the detection of the mucin protein CA19-9), which demonstrate that our SERS assay outperforms conventional assays (i.e., RIA and ELISA) with respect to limits of detection, readout time, and required sample volume.

Recent Trends in Antibody-based Oncologic Imaging

Antibodies, with their unmatched ability for selective binding to any target, are considered as potentially the most specific probes for imaging. Their clinical utility, however, has been limited chiefly due to their slow clearance from the circulation, longer retention in non-targeted tissues and the extensive optimization required for each antibody-tracer. The development of newer contrast agents, combined with improved conjugation strategies and novel engineered forms of antibodies (diabodies, minibodies, single chain variable fragments, and nanobodies), have triggered a new wave of antibody-based imaging approaches. Apart from their conventional use with nuclear imaging probes, antibodies and their modified forms are increasingly being employed with non-radioisotopic contrast agents (MRI and ultrasound) as well as newer imaging modalities, such as quantum dots, near infra red (NIR) probes, nanoshells and surface enhanced Raman spectroscopy (SERS). The review article discusses new developments in the usage of antibodies and their modified forms in conjunction with probes of various imaging modalities such as nuclear imaging, optical imaging, ultrasound, MRI, SERS and nanoshells in preclinical and clinical studies on the diagnosis, prognosis and therapeutic responses of cancer.

Mucins in pancreatic cancer and its microenvironment.

Pancreatic cancer remains a lethal malignancy with poor prognosis owing to therapeutic resistance, frequent recurrence and the absence of treatment strategies that specifically target the tumour and its supporting stroma. Deregulated cell-surface proteins drive neoplastic transformations and are envisioned to mediate crosstalk between the tumour and its microenvironment. Emerging studies have elaborated on the role of mucins in diverse biological functions, including enhanced tumorigenicity, invasiveness, metastasis and drug resistance through their characteristic O-linked and N-linked oligosaccharides (glycans), extended structures and unique domains. Multiple mucin domains differentially interact and regulate different components of the tumour microenvironment. This Review discusses: the expression pattern of various mucins in the pancreas under healthy, inflammatory, and cancerous conditions; the context-dependent attributes of mucins that differ under healthy and pathological conditions; the contribution of the tumour microenvironment in pancreatic cancer development and/or progression; diagnostic and/or prognostic efficacy of mucins; and mucin-based therapeutic strategies. Overall, this information should help to delineate the intricacies of pancreatic cancer by exploring the family of mucins, which, through various mechanisms in both tumour cells and the microenvironment, worsen disease outcome.

Early diagnosis of pancreatic cancer: challenges and new developments.

Pancreatic cancer is a lethal malignancy with its incidence almost equivalent to mortality. The complex pathophysiology, absence of early diagnostic and prognostic markers and unresponsiveness to radiation and chemotherapies are major barriers against successful therapy. Poor performance of therapeutic agents, even in the initial stage of invasive cases, emphasizes the importance of early detection for improved survival. The present review discusses the challenges and advances in biomarkers including serological signatures, circulating tumor cells, autoantibodies, epigenetic markers and miRNAs that are being explored to detect this cancer at early stages. Considering the long time gap between the development of malignant lesions and full-blown primary and metastatic pancreatic cancer, unique opportunities are being contemplated for the development of potential diagnostic and prognostic markers.

Nicotine/cigarette smoke promotes metastasis of pancreatic cancer through α7nAChR-mediated MUC4 upregulation

Despite evidence that long-term smoking is the leading risk factor for pancreatic malignancies, the underlying mechanism(s) for cigarette-smoke (CS)-induced pancreatic cancer (PC) pathogenesis has not been well established. Our previous studies revealed an aberrant expression of the MUC4 mucin in PC as compared with the normal pancreas, and its association with cancer progression and metastasis. Interestingly, here we explore a potential link between MUC4 expression and smoking-mediated PC pathogenesis and report that both cigarette smoke extract and nicotine, which is the major component of CS, significantly upregulates MUC4 in PC cells. This nicotine-mediated MUC4 overexpression was via the α7 subunit of nicotinic acetylcholine receptor (nAChR) stimulation and subsequent activation of the JAK2/STAT3 downstream signaling cascade in cooperation with the MEK/ERK1/2 pathway; this effect was blocked by the α7nAChR antagonists, α-bungarotoxin and mecamylamine, and by specific siRNA-mediated STAT3 inhibition. In addition, we demonstrated that nicotine-mediated MUC4 upregulation promotes the PC cell migration through the activation of the downstream effectors, such as HER2, c-Src and FAK; this effect was attenuated by shRNA-mediated MUC4 abrogation, further implying that these nicotine-mediated pathological effects on PC cells are MUC4 dependent. Furthermore, the in vivo studies showed a marked increase in the mean pancreatic tumor weight (low dose (100 mg/m3 total suspended particulate (TSP)), P=0.014; high dose (247 mg/m3 TSP), P=0.02) and significant tumor metastasis to various distant organs in the CS-exposed mice, orthotopically implanted with luciferase-transfected PC cells, as compared with the sham controls. Moreover, the CS-exposed mice had elevated levels of serum cotinine (low dose, 155.88±35.96 ng/ml; high dose, 216.25±29.95 ng/ml) and increased MUC4, α7nAChR and pSTAT3 expression in the pancreatic tumor tissues. Altogether, our findings revealed for the first time that CS upregulates the MUC4 mucin in PC via the α7nAChR/JAK2/STAT3 downstream signaling cascade, thereby promoting metastasis of PC.

Pancreatic cancer cells resistance to gemcitabine: The role of MUC4 mucin

Background:A major obstacle to the successful management of pancreatic cancer is to acquire resistance to the existing chemotherapeutic agents. Resistance to gemcitabine, the standard first-line chemotherapeutic agent for advanced and metastatic pancreatic cancer, is mainly attributed to an altered apoptotic threshold in the pancreatic cancer. The MUC4 transmembrane glycoprotein is aberrantly overexpressed in the pancreatic cancer and recently, has been shown to increase pancreatic tumour cell growth by the inhibition of apoptosis.Methods:Effect of MUC4 on pancreatic cancer cells resistance to gemcitabine was studied in MUC4-expressing and MUC4-knocked down pancreatic cancer cell lines after treatment with gemcitabine by Annexin-V staining, DNA fragmentation assay, assessment of mitochondrial cytochrome c release, immunoblotting and co-immunoprecipitation techniques.Results:Annexin-V staining and DNA fragmentation experiment demonstrated that MUC4 protects CD18/HPAF pancreatic cancer cells from gemcitabine-induced apoptosis. In concert with these results, MUC4 also attenuated mitochondrial cytochrome c release and the activation of caspase-9. Further, our results showed that MUC4 exerts anti-apoptotic function through HER2/extracellular signal-regulated kinase-dependent phosphorylation and inactivation of the pro-apoptotic protein Bad.Conclusion:Our results elucidate the function of MUC4 in imparting resistance to pancreatic cancer cells against gemcitabine through the activation of anti-apoptotic pathways and, thereby, promoting cell survival.

MUC4 potentiates invasion and metastasis of pancreatic cancer cells through stabilization of fibroblast growth factor receptor 1

MUC4 is a type-1 transmembrane mucin differentially expressed in multiple cancers and has previously been shown to potentiate progression and metastasis of pancreatic cancer. In this study, we investigated the molecular mechanisms associated with the MUC4-induced invasion and metastasis in pancreatic cancer. Stable silencing of MUC4 in multiple pancreatic cancer cells resulted in the downregulation of N-cadherin and its interacting partner fibroblast growth factor receptor 1 (FGFR1) through downregulation of partly by pFAK, pMKK7, pJNK and pc-Jun pathway and partly through PI-3K/Akt pathway. The downregulation of FGFR1 in turn led to downregulation of pAkt, pERK1/2, pNF-κB, pIkBα, uPA, MMP-9, vimentin, N-cadherin, Twist, Slug and Zeb1 and upregulation of E-cadherin, Occludin, Cytokeratin-18 and Caspase-9 in MUC4 knockdown BXPC3 and Capan1 cells compared with scramble vector transfected cells. Further, downregulation of FGFR1 was associated with a significant change in morphology and reorganization of the actin-cytoskeleton, leading to a significant decrease in motility (P < 0.00001) and invasion (P < 0.0001) in vitro and decreased tumorigenicity and incidence of metastasis in vivo upon orthotopic implantation in the athymic mice. Taken together, the results of the present study suggest that MUC4 promotes invasion and metastasis by FGFR1 stabilization through the N-cadherin upregulation.