Molecular basis for the interplay of apoptosis and proliferation mediated by Bcl-xL:Bim interactions in pancreatic cancer cells

A major mechanism through which cancer cells avoid apoptosis is by promoting the association of anti-apoptotic members of the pro-survival Bcl-2 protein family (like Bcl-2 and Bcl-xL) with BH(3) domain-only proteins (like Bim and Bid). Apoptosis and cell proliferation have been shown to be linked for many cancers but the molecular basis for this link is far from understood. We have identified the Bcl-xL:Bim protein-protein interface as a direct regulator of proliferation and apoptosis in pancreatic cancer cells. We were able to predict and subsequently verify experimentally the effect of various Bcl-xL single-point mutants (at the position A142) on binding to Bim by structural analysis and computational modeling of the inter-residue interactions at the Bcl-xL:Bim protein-protein interface. The mutants A142N, A142Q, and A142Y decreased binding of Bim to Bcl-xL and A142S increased this binding. The Bcl-xL mutants, with decreased affinity for Bim, caused an increase in apoptosis and a corresponding decrease in cell proliferation. However, we could prevent these effects by introducing a small interfering RNA (siRNA) targeted at Bim. These results show a novel role played by the Bcl-xL:Bim interaction in regulating proliferation of pancreatic cancer cells at the expense of apoptosis. This study presents a physiologically relevant model of the Bcl-xL:Bim interface that can be used for rational therapeutic design for the inhibition of proliferation and cancer cell resistance to apoptosis.     

Interpreting epithelial cancer biology in the context of stem cells: tumor properties and therapeutic implications

Over 90% of all human neoplasia is derived from epithelia. Significant progress has been made in the identification of stem cells of many epithelia. In general, epithelial stem cells lack differentiation markers, have superior in vivo and in vitro proliferative potential, form clusters in association with a specialized mesenchymal environment (the 'niche'), are located in well-protected and nourished sites, and are slow-cycling and thus can be experimentally identified as 'label-retaining cells'. Stem cells may divide symmetrically giving rise to two identical stem cell progeny. Any stem cells in the niche, which defines the size of the stem cell pool, may be randomly expelled from the niche due to population pressure (the stochastic model). Alternatively, a stem cell may divide asymmetrically yielding one stem cell and one non-stem cell that is destined to exit from the stem cell niche (asymmetric division model). Stem cells separated from their niche lose their stemness, although such a loss may be reversible, becoming 'transit-amplifying cells' that are rapidly proliferating but have a more limited proliferative potential, and can give rise to terminally differentiated cells. The identification of the stem cell subpopulation in a normal epithelium leads to a better understanding of many previously enigmatic properties of an epithelium including the preferential sites of carcinoma formation, as exemplified by the almost exclusive association of corneal epithelial carcinoma with the limbus, the corneal epithelial stem cell zone. Being long-term residents in an epithelium, stem cells are uniquely susceptible to the accumulation of multiple, oncogenic changes giving rise to tumors. The application of the stem cell concept can explain many important carcinoma features including the clonal origin and heterogeneity of tumors, the occasional formation of tumors from the transit amplifying cells or progenitor cells, the formation of precancerous 'patches' and 'fields', the mesenchymal influence on carcinoma formation and behavior, and the plasticity of tumor cells. While the concept of cancer stem cells is extremely useful and it is generally assumed that such cells are derived from normal stem cells, more work is needed to identify and characterize epithelial cancer stem cells, to address their precise relationship with normal stem cells, to study their markers and their proliferative and differentiation properties and to design new therapies that can overcome their unusual resistance to chemotherapy and other conventional tumor modalities.     

Scarless integumentary wound healing in the mammalian fetus: Molecular basis and therapeutic implications

Adult mammals respond to injury of their skin/integument by forming scar tissue. Scar is useful in rapidly sealing an injured area, but can also lead to significant morbidity. Mammals in fetal life retain the ability to heal integumentary wounds regeneratively, without scar. The critical molecular mechanisms governing this remarkable phenomenon have been a subject of great interest, in the hopes that these could be dissected and recapitulated in the healing adult wound, with the goal of inducing scarless healing in injured patients. Multiple lines of investigation spanning decades have implicated a number of factors in distinguishing scarless from fibrotic wound healing, including most prominently transforming growth factor‐β and interleukin‐10, among others. Therapeutic interventions to try to mitigate scarring in adult wounds have been developed out of these studies, and have reached the level of clinical trials in humans, although as yet no FDA‐approved treatment exists. More recent expressomic studies have revealed many more genes that are differentially expressed in scarlessly healing fetal wounds compared with adult, and microRNAs have also been identified as participating in the fetal wound healing response. These represent an even greater range of potential therapeutics (or targets for therapy) to translate the promise of scarless fetal wound healing to the injured adult patient. Birth Defects Research (Part C) 96:223–236, 2012. © 2012 Wiley Periodicals, Inc.     

Human pancreatic cancer stem cells: implications for how we treat pancreatic cancer

Pancreatic cancer has the worst prognosis of any major malignancy, with an annual death rate that approximates the annual incidence rate. Delayed diagnosis, relative chemotherapy and radiation resistance and an intrinsic biologic aggressiveness all contribute to the abysmal prognosis associated with pancreatic cancer. Answers to the frustrating effort to find effective therapies for pancreatic cancer may be gained through a renewed perspective on tumorigenesis as a process governed by a select population of cells, termed cancer stem cells (CSCs). Cancer stem cells, like their normal counterparts, have the properties of self-renewal and multilineage differentiation and possess inherently heightened DNA damage response and repair mechanisms that make them difficult to eradicate. Initially discovered in leukemias, researchers have identified CSCs in several solid-organ malignancies including breast, brain, prostate, and colon cancers. We have recently identified a CSC population in human pancreatic cancers. These pancreatic CSC represent 0.5% to 1.0% of all pancreatic cancer cells and express the cell surface markers CD44, CD24, and epithelial-specific antigen. Pancreatic CSCs have been shown to be resistant to standard chemotherapy and radiation, and devising specific therapies to target this distinct cell population is likely needed to identify effective therapies to treat this dismal disease.     

Molecular basis of inherited skin-blistering disorders, and therapeutic implications

Epidermolysis bullosa (EB) and associated skin-fragility syndromes are a group of inherited skin diseases characterised by trauma-induced blistering of the skin and mucous membranes. Mutations in at least 14 distinct genes encoding molecular components of the epidermis or the dermal-epidermal junction (DEJ) can cause blistering skin diseases that differ by clinical presentation and severity of the symptoms. Despite great advances in discerning the genetic basis of this group of diseases, the molecular pathways leading to symptoms are not yet fully understood. Unravelling these pathways by molecular analysis of the structure and in vitro assessment of functional properties of the human proteins involved, combined with genetic models in lower organisms, should pave the way for specific cures for inherited skin fragility.     

Glioblastoma stem cells:Molecular characteristics and therapeutic implications

Glioblastoma Multiforme(GBM)is a grade IV astrocytoma,with a median survival of 14.6 mo.Within GBM,stem-like cells,namely glioblastoma stem cells(GSCs),have the ability to self-renew,differentiate into distinct lineages within the tumor and initiate tumor xenografts in immunocompromised animal models.More importantly,GSCs utilize cell-autonomous and tumor microenvironment-mediated mechanisms to overcome current therapeutic approaches.They are,therefore,very important therapeutic targets.Although the functional criteria defining GSCs are well defined,their molecular characteristics,the mechanisms whereby they establish the cellular hierarchy within tumors,and their contribution to tumor heterogeneity are not well understood.This review is aimed at summarizing current findings about GSCs and their therapeutic importance from a molecular and cellular point of view.A better characterization of GSCs is crucial for designing effective GSCtargeted therapies.     

The molecular basis of lung cancer: molecular abnormalities and therapeutic implications

Lung cancer is the number one cause of cancer-related death in the western world. Its incidence is highly correlated with cigarette smoking, and about 10% of long-term smokers will eventually be diagnosed with lung cancer, underscoring the need for strengthened anti-tobacco policies. Among the 10% of patients who develop lung cancer without a smoking history, the environmental or inherited causes of lung cancer are usually unclear. There is no validated screening method for lung cancer even in high-risk populations and the overall five-year survival has not changed significantly in the last 20 years. However, major progress has been made in the understanding of the disease and we are beginning to see this knowledge translated into the clinic. In this review, we will summarize the current state of knowledge regarding the cascade of events associated with lung cancer development. From subclinical DNA damage to overt invasive disease, the mechanisms leading to clinically and molecularly heterogeneous tumors are being unraveled. These lesions allow cells to escape the normal regulation of cell division, apoptosis and invasion. While all subtypes of non-small cell lung cancer have historically been treated the same, stage-for-stage, recent technological advances have allowed a better understanding of the molecular classification of the disease and provide hypotheses for molecular early detection and targeted therapeutic strategies.     

Targeted destruction of prostate cancer cells and xenografts by lytic peptide-betaLH conjugates

In series of experiments conducted in vitro, we have established the concept that conjugates of the lytic peptides Hecate or Phor14 with a fragment of the beta chain of LH (amino acids 80-94) selectively destroy both androgen sensitive and insensitive human prostate cancer cells. Extraction of steroids from the culture medium by charcoal reduced the ability of the conjugates to kill LNCaP, BRF41T and PC-3 cells. Addition of hormones known to up-regulate LH receptors (estradiol, testosterone or FSH) to the culture medium restored the ability of the conjugates to kill these cell lines. The toxicity of the conjugates (EC(50)) to these cell lines was closely correlated to their LH binding capacities (f mol/10(6) cells). In series of in vivo experiments we have shown that both the Hecate and Phor14-betaLH conjugates are remarkably effective in causing tumor cell necrosis and cessation of tumor growth in nude athymic mice. Treatment with Hecate-betaLH (12 mg/kg body weight) resulted in a reduction of tumor burden (mg tumor/g body weight) from 60 to 14 (P<0.0001); treatment with Phor14-betaLH (12 mg/kg body weight) reduced tumor burden to 27 mg (P<0.0001). Treatment with a high dose of Phor14-betaLH (24 mg/kg body weight) reduced the tumor burden from 60 to 12 mg/kg P<0.0001). Pretreatment of animals receiving a low dose of Phor14-betaLH (12 mg/kg) with either estradiol or follicle stimulating hormone, (FSH) resulted in reduction of tumor burden from 60 to 11 mg/kg. Administration of a second 3-week treatment after a one month recovery period caused complete regression of more than 75 percent of the tumors. No changes in body weight or histological abnormalities were found in any of the organs examined, except the testes.     

Killer dendritic cells: mechanisms of action and therapeutic implications for cancer

Dendritic cells (DC) are essential for the development and regulation of adaptive host immune responses against tumors. DC are heterogeneous and comprised of diverse cellular subsets. They are best known for mediating a crucial role in the initiation of acquired immunity by serving as professional antigen presenting cells (APC) that take up antigens in their local microenvironment, which are then processed and presented to na?ve T cells in the context of major histocompatibility complex (MHC) class I and II molecules. In addition to these functions, DC can modulate the types of T cell responses they generate, and can also influence the responses of innate effectors, such as NK cells. There is also now evidence that they may mediate a more primordial role as innate, effector cells that are tumoricidal. 'Killer' DC (KDC) may represent a true 'multi-tasking' cell type that can sequentially act as a 'hunter-gatherer' of antigens; as well as, an instructor, then enforcer/regulator, of antigen-specific anti-tumor T-cell responses in vivo. In this review, we will critically examine the published record regarding KDC, their mechanism(s) of action, and then consider the potential integration of KDC into novel immunotherapies for patients with cancer.     

Molecular basis of potassium channels in pancreatic duct epithelial cells

Potassium channels regulate excitability, epithelial ion transport, proliferation, and apoptosis. In pancreatic ducts, K⁺ channels hyperpolarize the membrane potential and provide the driving force for anion secretion. This review focuses on the molecular candidates of functional K⁺ channels in pancreatic duct cells, including KCNN4 (K_Ca3.1), KCNMA1 (K_Ca1.1), KCNQ1 (K_v7.1), KCNH2 (K_v11.1), KCNH5 (K_v10.2), KCNT1 (K_Ca4.1), KCNT2 (K_Ca4.2), and KCNK5 (K_2P5.1). We will give an overview of K⁺ channels with respect to their electrophysiological and pharmacological characteristics and regulation, which we know from other cell types, preferably in epithelia, and, where known, their identification and functions in pancreatic ducts and in adenocarcinoma cells. We conclude by pointing out some outstanding questions and future directions in pancreatic K⁺ channel research with respect to the physiology of secretion and pancreatic pathologies, including pancreatitis, cystic fibrosis, and cancer, in which the dysregulation or altered expression of K⁺ channels may be of importance.     

Autophagic tumor stroma: A biofuel for cancer growth

Comment on: Castello-Cros R, et al. Cell Cycle 2011; 10:2021-34.     

Involvement of host stroma cells and tissue fibrosis in pancreatic tumor development in transgenic mice

Introduction

Stroma cells and extracellular matrix (ECM) components provide the pivotal microenvironment for tumor development. The study aimed to evaluate the importance of the pancreatic stroma for tumor development.

Methods

Pancreatic tumor cells were implanted subcutaneously into green fluorescent protein transgenic mice, and stroma cells invading the tumors were identified through immunohistochemistry. Inhibition of tumor invasion by stroma cells was achieved with halofuginone, an inhibitor of TGFβ/Smad3 signaling, alone or in combination with chemotherapy. The origin of tumor ECM was evaluated with species-specific collagen I antibodies and in situ hybridization of collagen α1(I) gene. Pancreatic fibrosis was induced by cerulean injection and tumors by spleen injection of pancreatic tumor cells.

Results

Inhibition of stroma cell infiltration and reduction of tumor ECM levels by halofuginone inhibited development of tumors derived from mouse and human pancreatic cancer cells. Halofuginone reduced the number only of stroma myofibroblasts expressing both contractile and collagen biosynthesis markers. Both stroma myofibroblasts and tumor cells generated ECM that contributes to tumor growth. Combination of treatments that inhibit stroma cell infiltration, cause apoptosis of myofibroblasts and inhibit Smad3 phosphorylation, with chemotherapy that increases tumor-cell apoptosis without affecting Smad3 phosphorylation was more efficacious than either treatment alone. More tumors developed in fibrotic than in normal pancreas, and prevention of tissue fibrosis greatly reduced tumor development.

Conclusions

The utmost importance of tissue fibrosis and of stroma cells for tumor development presents potential new therapy targets, suggesting combination therapy against stroma and neoplastic cells as a treatment of choice.     

Hallmarks of cancer: Interactions with the tumor stroma

Ten years ago, Hanahan and Weinberg delineated six “Hallmarks of cancer” which summarize several decades of intense cancer research. However, tumor cells do not act in isolation, but rather subsist in a rich microenvironment provided by resident fibroblasts, endothelial cells, pericytes, leukocytes, and extra-cellular matrix. It is increasingly appreciated that the tumor stroma is an integral part of cancer initiation, growth and progression. The stromal elements of tumors hold prognostic, as well as response-predictive, information, and abundant targeting opportunities within the tumor microenvironment are continually identified. Herein we review the current understanding of tumor cell interactions with the tumor stroma with a particular focus on cancer-associated fibroblasts and pericytes. Moreover, we discuss emerging fields of research which need to be further explored in order to fulfil the promise of stroma-targeted therapies for cancer.     

Cellular sources of new pancreatic beta cells and therapeutic implications for regenerative medicine

Replacing missing insulin-producing beta cells to treat diabetes is a major challenge for regenerative medicine. A better understanding of beta-cell embryogenesis and regeneration in adult life is needed to devise means to derive these specialized cells in sufficiently large numbers from stem or precursor cells. It is also critical to ensure that any surrogate or regenerated beta cells have perfectly regulated insulin production, which is essential for physiological glucose homeostasis.     

Tumor microenvironment: the role of the tumor stroma in cancer

The tumor microenvironment, composed of non-cancer cells and their stroma, has become recognized as a major factor influencing the growth of cancer. The microenvironment has been implicated in the regulation of cell growth, determining metastatic potential and possibly determining location of metastatic disease, and impacting the outcome of therapy. While the stromal cells are not malignant per se, their role in supporting cancer growth is so vital to the survival of the tumor that they have become an attractive target for chemotherapeutic agents. In this review, we will discuss the various cellular and molecular components of the stromal environment, their effects on cancer cell dynamics, and the rationale and implications of targeting this environment for control of cancer. Additionally, we will emphasize the role of the bone marrow-derived cell in providing cells for the stroma.     

Circulating tumor cells in the diagnosis and management of pancreatic cancer

Pancreatic cancers are typically resistant to chemo and radiation therapy and are predisposed to distant metastases. Circulating tumor cells (CTCs) are tumor cells disseminated from primary and metastatic sites and can be isolated from peripheral blood. CTC may overcome the limitation of the current available tumor markers, CA19-9. As a surrogate for 'real-time biopsy', CTCs allow recurrent assessment of a tumor's biological activity. We review the current methodologies for CTC extraction and characterization including antibody-based immunological assays, PCR-based assays, and novel technologies based on the physical or biological characteristics of CTCs. CTCs also provide an accessible link to the existence of epithelial to mesenchymal transition, tumor stem cell markers, and ongoing clonal mutations and epigenetic changes in the tumor. We also explore the potential of using CTC profiling in diagnosis, selection of neoadjuvant and adjuvant therapy, detection of recurrent disease, examination of pharmacodynamic biomarkers, as well as in gene therapy and immunotherapy for pancreatic cancer. Ongoing CTC characterization not only has the potential to represent all cells shed from primary pancreatic tumor and each metastatic site, but also allows dynamic sampling at multiple time points during the clinical course to identify the subpopulations of CTCs and the specific molecules driving metastasis and chemo resistance. We predict that CTC genotyping and phenotyping will play an increasing role in personalized therapy and in identification of novel therapeutic targets as well as monitoring the course and status of the disease.     

Chronic tendon pathology: molecular basis and therapeutic implications

Tendons are frequently affected by chronic pain or rupture. Many causative factors have been implicated in the pathology, which until relatively recently was under-researched and poorly understood. There is now a greater knowledge of the molecular basis of tendon disease. Most tendon pathology (tendinopathy) is associated with degeneration, which is thought to be an active, cell-mediated process involving increased turnover and remodelling of the tendon extracellular matrix. Degradation of the tendon matrix is mediated by a variety of metalloproteinase enzymes, including matrix metalloproteinases and 'aggrecanases'. Neuropeptides and other factors released by stimulated cells or nerve endings in or around the tendon might influence matrix turnover, and could provide novel targets for therapeutic intervention.