Proteomic analysis of MCF-7 breast cancer cell line exposed to mitogenic concentration of 17beta-estradiol

Estrogens are powerful mitogens that play a critical role in the onset of breast cancer and its progression. About two-thirds of all breast cancers are estrogen receptor (ER)+ at the time of diagnosis, and the ER expression is the determinant of a tumor phenotype associated with hormone responsiveness. The molecular basis of the relationship between ER expression, (anti)hormonal responsiveness, and breast cancer prognosis is still unknown. To identify the proteins affected by the presence of the hormone we used 2-D-PAGE-based bottom-up proteomics for the study of the proteome of MCF-7 cells of estrogen-responsive breast carcinoma exposed to a mitogenic concentration of 17beta-estradiol (E2) for 12, 18, 24, and 30 h. Differential expression analysis showed significant changes for 12 proteins. These include ezrin-radixin-moesin-binding phosphoprotein of 50 kDa which was previously shown to be directly regulated by E2. Expression profiles of other proteins already implicated in the progression of breast cancer, such as stathmin, calreticulin, heat shock 71 kDa, alpha-enolase are also described. Moreover, it is observed that different unexpected proteins, translation factors, and energetic metabolism enzymes are also influenced by the presence of the hormone.     

Human placental trophoblast as an in vitro model for tumor progression

The human placenta is a highly invasive tumor-like structure in which a subpopulation of placental trophoblast cells known as the "extravillous trophoblast" (EVT) invades the uterine decidua and its vasculature to establish adequate fetal-maternal exchange of molecules. By utilizing in vitro-propagated short-lived EVT cell lines we found that molecular mechanisms responsible for their invasiveness are identical to those of cancer cells; however, unlike cancer cells, their proliferation, migration, and invasiveness in situ are stringently controlled by decidua-derived transforming growth factor (TGF)-beta. By SV40T antigen transfection of normal EVT cells followed by a forced crisis regimen in culture we produced an immortalized premalignant derivative that is hyperproliferative, hyperinvasive, and deficient in gap-junctional intercellular communication. Both premalignant and malignant EVT (JAR and JEG-3 choriocarcinoma) cell lines were found to be TGF-beta-resistant. Using these cell lines, we investigated genetic changes responsible for transition of the normal EVT cells to premalignant and malignant phenotype. Hyperinvasiveness in both cases resulted from a downregulation of tissue inhibitor of metalloprotease (TIMP)-1 and plasminogen activator inhibitor (PAI)-1 genes. In contrast to normal EVT cells, both cell types failed to upregulate these genes in response to TGF-beta. Loss of TGF-beta response in malignant EVT cells was explained by the loss of expression of Smad3 gene. Differential mRNA display of normal and premalignant EVT cells identified up- and down-regulation of numerous known or novel genes in premalignant EVT cells, with potential oncogenic and (or) tumor-suppressor functions, e.g., loss of fibronectin and insulin-like growth factor binding protein (IGFBP-5). Premalignant EVT cells also lost IGF receptor type 2 (IGFR-II). IGFBP-5 was shown to be a negative regulator of IGF-1-induced proliferation of premalignant EVT cells, so that loss of IGFBP-5 as well as IGFR-II permitted their unrestricted proliferation in an IGF-I-rich microenvironment of the fetal-maternal interface. The present model may be a good prototype for identifying genetic changes underlying epithelial tumor progression.     

Calcium and calcium-related proteins in endometrial cancer: opportunities for pharmacological intervention

Intracellular calcium ions are key second messengers and play an important role in malignant transformation and cancer progression. Estrogen can evoke intracellular calcium increases through membrane-initiated effects and activate subsequent kinase cascades within minutes in normal and cancerous epithelial cells. Ca²⁺-related proteins are expressed in normal epithelial cells or endometrial cancer cells, some of which are upregulated by estrogen. Both estrogen-induced transient calcium increases and long-term changes in protein expression levels may be involved in regulating cancer initiation, progression and metastasis. Calcium channel blockers are reported to regulate both the rapid estrogen-induced intracellular Ca²⁺ increase and cell proliferation, apoptosis and migration, thus having the potential for pharmacological modulators to be repurposed for the treatment of endometrial cancer.     

Semaphorins in cancer: Biological mechanisms and therapeutic approaches

The hallmarks of cancer include multiple alterations in the physiological processes occurring in normal tissues, such as cell proliferation, apoptosis, and restricted cell migration. These aberrant behaviors are due to genetic and epigenetic changes that affect signaling pathways controlling cancer cells, as well as the surrounding “normal” cells in the tumor microenvironment. Semaphorins and their receptors (mainly plexins and neuropilins) are aberrantly expressed in human tumors, and multiple family members are emerging as pivotal signals deregulated in cancer. Notably, different semaphorins can promote or inhibit tumor progression, depending on the implicated receptor complexes and responsive cell type. The important role of semaphorin signals in the regulation of tumor angiogenesis, invasion and metastasis has initiated multiple experimental approaches aimed at targeting these pathways to inhibit cancer.     

Collective dynamics of cancer cells confined in a confluent monolayer of normal cells

Tumorigenesis often involves specific changes in cell motility and intercellular adhesion. Understanding the collective cancer cell behavior associated with these specific changes could facilitate the detection of malignant characteristics during tumor growth and invasion. In this study, a cellular vertex model is developed to investigate the collective dynamics of a disk-like aggregate of cancer cells confined in a confluent monolayer of normal cells. The effects of intercellular adhesion and cell motility on tumor progression are examined. It is found that the stresses in both the cancer cells and the normal cells increase with tumor growth, resulting in a crowded environment and enhanced cell apoptosis. The intercellular adhesion between cancer cells and normal cells is revealed to promote tumor growth and invasion. The tumor invasion dynamics hinges on the motility of cancer cells. The cancer cells could orchestrate into different collective migration modes, e.g., directional migration and rotational oscillations, dictated by the competition between cell persistence and local coordination. Phase diagrams are established to reveal the competitive mechanisms. This work highlights the role of mechanics in regulating tumor growth and invasion.     

Metabolomic Changes Accompanying Transformation and Acquisition of Metastatic Potential in a Syngeneic Mouse Mammary Tumor Model

Breast cancer is the most common cancer type for women in the western world. Despite decades of research, the molecular processes associated with breast cancer progression are still inadequately defined. Here, we focus on the systematic alteration of metabolism by using the state of the art metabolomic profiling techniques to investigate the changes of 157 metabolites during the progression of normal mouse mammary epithelial cells to an isogenic series of mammary tumor cell lines with increasing metastatic potentials. Our results suggest a two-step metabolic progression hypothesis during the acquisition of tumorigenic and metastatic abilities. Metabolite changes accompanying tumor progression are identified in the intracellular and secreted forms in several pathways, including glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, fatty acid and nucleotide biosynthesis, and the GSH-dependent antioxidative pathway. These results suggest possible biomarkers of breast cancer progression as well as opportunities of interrupting tumor progression through the targeting of metabolic pathways.     

Are cancer cells really softer than normal cells?

Solid tumours are often first diagnosed by palpation, suggesting that the tumour is more rigid than its surrounding environment. Paradoxically, individual cancer cells appear to be softer than their healthy counterparts. In this review, we first list the physiological reasons indicating that cancer cells may be more deformable than normal cells. Next, we describe the biophysical tools that have been developed in recent years to characterise and model cancer cell mechanics. By reviewing the experimental studies that compared the mechanics of individual normal and cancer cells, we argue that cancer cells can indeed be considered as softer than normal cells. We then focus on the intracellular elements that could be responsible for the softening of cancer cells. Finally, we ask whether the mechanical differences between normal and cancer cells can be used as diagnostic or prognostic markers of cancer progression.     

Why cancer and inflammation?

Central to the development of cancer are genetic changes that endow these “cancer cells” with many of the hallmarks of cancer, such as self-sufficient growth and resistance to anti-growth and pro-death signals. However, while the genetic changes that occur within cancer cells themselves, such as activated oncogenes or dysfunctional tumor suppressors, are responsible for many aspects of cancer development, they are not sufficient. Tumor promotion and progression are dependent on ancillary processes provided by cells of the tumor environment but that are not necessarily cancerous themselves. Inflammation has long been associated with the development of cancer. This review will discuss the reflexive relationship between cancer and inflammation with particular focus on how considering the role of inflammation in physiologic processes such as the maintenance of tissue homeostasis and repair may provide a logical framework for understanding the connection between the inflammatory response and cancer.     

Facts and theories concerning the mechanisms of carcinogenesis

Carcinogenesis can be induced experimentally by exposure to exogenous agents or it can occur spontaneously without intentional or active intervention. Carcinogenesis can be actively induced by chemicals, radiation, infectious biological agents, transgenesis, or selective breeding. In the human and occasionally when testing potential carcinogens in animals, cancer may result from passive exposure to carcinogens encountered in the ambient environment or from changes in the internal milieu of the animal. Many carcinogens alter the structure of DNA resulting in carcinogenesis, but a significant number of carcinogens do not appear to act through this mechanism. When the action of specific carcinogenic agents is considered in relation to the stages of cancer development, initiation, promotion, and progression, the mechanism of the induction of carcinogenesis by DNA-reactive agents that alter genomic structure can be reconciled with those agents that do not act in this manner. As some cells are fortuitously initiated by uncontrolled variables such as irradiation and through changes in normal processes, the stimulation of growth and altered genetic expression by nongenotoxic agents may result indirectly in cancer development. The final stage of carcinogenesis, progression, can occur spontaneously, enhanced by formation and propagation of genetic errors due to increased cellular proliferation associated with the promotion stage. In addition, chemical and viral agents that lack the capacity for initiation and promotion may actively convert cells in the stage of promotion to the stage of progression. Therefore, the diverse mechanisms of action of carcinogenic agents in relation to their effects on specific stages in the natural history of cancer development allow for greater congruence of many of the theories of carcinogenesis. The influence of the roles of nongenotoxic carcinogenic agents and the potential role of progressor agents on the carcinogenesis process allow a more accurate identification of the potential risk that specific carcinogenic agents pose for increasing human cancer.     

17 beta-hydroxysteroid dehydrogenases and cancers

17β-Hydroxysteroid dehydrogenases (17HSDs) catalyze the interconversions between active 17β-hydroxysteroids and less-active 17-ketosteroids thereby affecting the availability of biologically active estrogens and androgens in a variety of tissues. The enzymes have different enzymatic properties and characteristic cell-specific expression patterns, suggesting differential physiological functions for the enzymes. Epidemiological and endocrine evidence indicate that estrogens play a key role in the etiology of breast cancer while androgens are involved in mechanisms controlling the growth of prostatic cells, both normal and malignant. Recently, we have developed, using LNCaP prostate cancer cell lines, a cell model to study the progression of prostate cancer. In the model LNCaP cells are transformed in culture condition to more aggressive cells, able to grow in suspension cultures. Our results suggest that substantial changes in androgen and estrogen metabolism occur in the cells during the process. These changes lead to increased production of active estrogens during transformation of the cells. Data from studies of breast cell lines and tissues suggest that the oxidative 17HSD type 2 may predominate in human non-malignant breast epithelial cells, while the reductive 17HSD type 1 activity prevails in malignant cells. Deprivation of an estrogen response by using specific 17HSD type 1 inhibitors is a tempting approach to treat estrogen-dependent breast cancer. Our recent studies demonstrate that in addition to sex hormone target tissues, estrogens may be important in the development of cancer in some other tissues previously not considered as estrogen target tissues such as colon. Our data show that the abundant expression of 17HSD type 2 present in normal colonic mucosa is significantly decreased during colon cancer development.     

Progression elevated gene-3, PEG-3, induces genomic instability in rodent and human tumor cells

Genomic instability is a fundamental component of cancer progression. Subtraction hybridization identified a novel rodent gene, progression elevated gene-3 (PEG-3) whose expression directly correlates with cancer aggressiveness and progression. Moreover, ectopic expression of PEG-3 in rodent or human tumor cells produces an aggressive transformed phenotype. We demonstrate that PEG-3 expression in rodent tumor cells correlates directly with genomic instability as characterized by alterations in chromosome composition and structure. Additionally, elevated endogenous or ectopic expression of PEG-3 in rodent and human tumor cells, respectively, enhances gene amplification, as monitored by resistance to methothrexate (MTX) and amplification of the dihydrofolate reductase (dhfr) gene. Stable expression of PEG-3 in normal cloned rat embryo fibroblast (CREF) cells marginally elevates MTX resistance, but morphology remains unaltered and anchorage independence is not induced, suggesting that these phenotypes are separable in immortal cells and gene amplification may precede the acquisition of morphological and oncogenic transformation. The present studies document that stable, inducible, and transient expression of PEG-3 in cancer cells augments genomic instability. In these contexts, one mechanism by which PEG-3 influences cancer progression may be by preferentially facilitating the development of genomic changes in evolving cancer cells.     

Reprogramming of human cancer cells to pluripotency for models of cancer progression

The ability to study live cells as they progress through the stages of cancer provides the opportunity to discover dynamic networks underlying pathology, markers of early stages, and ways to assess therapeutics. Genetically engineered animal models of cancer, where it is possible to study the consequences of temporal‐specific induction of oncogenes or deletion of tumor suppressors, have yielded major insights into cancer progression. Yet differences exist between animal and human cancers, such as in markers of progression and response to therapeutics. Thus, there is a need for human cell models of cancer progression. Most human cell models of cancer are based on tumor cell lines and xenografts of primary tumor cells that resemble the advanced tumor state, from which the cells were derived, and thus do not recapitulate disease progression. Yet a subset of cancer types have been reprogrammed to pluripotency or near‐pluripotency by blastocyst injection, by somatic cell nuclear transfer and by induced pluripotent stem cell (iPS) technology. The reprogrammed cancer cells show that pluripotency can transiently dominate over the cancer phenotype. Diverse studies show that reprogrammed cancer cells can, in some cases, exhibit early‐stage phenotypes reflective of only partial expression of the cancer genome. In one case, reprogrammed human pancreatic cancer cells have been shown to recapitulate stages of cancer progression, from early to late stages, thus providing a model for studying pancreatic cancer development in human cells where previously such could only be discerned from mouse models. We discuss these findings, the challenges in developing such models and their current limitations, and ways that iPS reprogramming may be enhanced to develop human cell models of cancer progression.     

Tumour-host interactions: implications for developing anti-cancer therapies

Cancers are a complex set of proliferative diseases that arise in most cases through multi-step pathways involving an accumulation of genetic and epigenetic changes. These steps include inactivation of tumour suppressor genes and activation of oncogenes. However, in addition to genetic mutations in the tumour cells themselves, the local host environment can act as a critical modulator of cancer progression, having either tumour-suppressive or tumour-promoting effects depending on the stage and site of cancer development. Because stromal cells can have these opposing functions during cancer development and progression, a recurring theme throughout this review will be that of balance: maintaining the normal functions of these co-opted cells, yet selectively inhibiting their pro-tumourigenic functions. To achieve this equilibrium, we need to understand the molecular mechanisms by which normal cells become modified by cancer cells before we can hope to target these functions selectively. Here, we will discuss recent efforts to address these key challenges and offer perspectives on the translation of discoveries made in model systems to the clinic.     

Spatiotemporal switching signals for cancer stem cell activation in pediatric origins of adulthood cancer:Towards a watch-and-wait lifetime strategy for cancer treatment

Pediatric origin of cancer stem cell hypothesis holds great promise and potential in adult cancer treatment, however; the road to innovation is full of obstacles as there are plenty of questions left unanswered. First, the key question is to characterize the nature of such stem cells (concept). Second, the quantitative imaging of pediatric stem cells should be implemented(technology). Conceptually, pediatric stem cell origins of adult cancer are based on the notion that plasticity in early life developmental programming evolves local environments to cancer. Technologically, such imaging in children is lacking as all imaging is designed for adult patients. We postulate that the need for quantitative imaging to measure space-time changes of plasticity in early life developmental programming in children may trigger research and development of the imaging technology. Such quantitative imaging of pediatric origin of adulthood cancer will help develop a spatiotemporal monitoring system to determine cancer initiation and progression. Clinical validation of such speculative hypothesis-that cancer originates in a pediatric environment-will help implement a wait-andwatch strategy for cancer treatment.