Epstein-Barr Virus-Positive Large Cell Neuroendocrine Carcinoma of the Nasopharynx: Report of a Case with Complete Clinical and Radiological Response After Combined Chemoradiotherapy.

Neuroendocrine carcinomas of the head and neck are rare and are classified as well differentiated, moderately differentiated, and poorly differentiated carcinomas with the latter category being subdivided into small cell and large cell neuroendocrine carcinoma (LCNEC). While most carcinomas in the nasopharynx are associated with Epstein-Barr virus (EBV), there has been only one previous report demonstrating a link between EBV and LCNEC of the nasopharynx. In this report we describe a second case of EBV-positive LCNEC arising in the nasopharynx with bilateral cervical metastases. The patient was treated with a combination of radiation and chemotherapy which resulted in a complete clinical and radiological response. The patient is still disease free 3 years after presentation. The results of this case suggest that EBV-positive LCNEC is sensitive to chemoradiotherapy and as a result may have better prognosis than EBV-negative LCNEC arising in the nasopharynx or other sites.

When thyroid carcinoma goes bad: a morphological and molecular analysis.

Thyroid carcinomas of follicular epithelial derivation are common and generally well-behaved malignancies with excellent cure and survival rates. However, a subset of these carcinomas, whether well-differentiated, poorly differentiated, or anaplastic, is highly aggressive, manifesting with local invasion, recurrence, and distant metastasis. The recognition of dedifferentiation is of paramount importance. In addition, the challenge for Pathologists is to identify the rare aggressive differentiated carcinomas so that treatment may be tailored appropriately. Thus, histological subtyping and documentation of other aggressive features such as widespread invasion and angioinvasion are critical. Mutational analyses in the past decade have delineated the molecular alterations responsible for thyroid carcinogenesis and progression, allowing greater sub-classification and prognostication of thyroid carcinomas. This review article highlights important aggressive morphologic features and molecular mutations associated with thyroid carcinoma.

Low-grade fibromyxoid sarcoma mimicking solitary fibrous tumor: a report of two cases.

Low-grade fibromyxoid sarcoma (LGFMS) is an uncommon fibroblastic neoplasm with many morphologic mimics. Solitary fibrous tumor is a more common fibroblastic neoplasm, but the two rarely enter the same differential diagnosis. However, here, we report two unusual cases of LGFMS, containing dilated, hemangiopericytoma-like blood vessels, which prompted diagnostic considerations of solitary fibrous tumor. Both cases presented were confirmed to harbor FUS gene rearrangement, thereby confirming a diagnosis of LGFMS. One case is that of an 18-year-old male with a left forearm mass, and the other a 50-year-old man with a left popliteal mass. While both cases show some histologic features of LGFMS, the non-classical, dilated blood vessel pattern seen here may serve as a diagnostic pitfall, as LGFMS normally exhibits fine, curvilinear blood vessels. To our knowledge, there is only one other report of LGFMS displaying such hemangiopericytoma-like blood vessels. In summary, when encountering a bland spindle cell neoplasm with classic hemangiopericytoma-like blood vessels, it is prudent to consider a diagnosis of LGFMS besides solitary fibrous tumor--particularly in the absence of CD34 immunoreactivity as it may be a rare, mimicking variant of LGFMS.

Calreticulin affects cell adhesiveness through differential phosphorylation of insulin receptor substrate-1.

Cellular adhesion to the underlying substratum is regulated through numerous signaling pathways. It has been suggested that insulin receptor substrate 1 (IRS-1) is involved in some of these pathways, via association with and activation of transmembrane integrins. Calreticulin, as an important endoplasmic reticulum-resident, calcium-binding protein with a chaperone function, plays an obvious role in proteomic expression. Our previous work showed that calreticulin mediates cell adhesion not only by affecting protein expression but also by affecting the state of regulatory protein phosphorylation, such as that of c-src. Here, we demonstrate that calreticulin affects the abundance of IRS-1 such that the absence of calreticulin is paralleled by a decrease in IRS-1 levels and the unregulated overexpression of calreticulin is accompanied by an increase in IRS-1 levels. These changes in the abundance of calreticulin and IRS-1 are accompanied by changes in cell-substratum adhesiveness and phosphorylation, such that increases in the expression of calreticulin and IRS-1 are paralleled by an increase in focal contact-based cell-substratum adhesiveness, and a decrease in the expression of these proteins brings about a decrease in cell-substratum adhesiveness. Wild type and calreticulin-null mouse embryonic fibroblasts (MEFs) were cultured and the IRS-1 isoform profile was assessed. Differences in morphology and motility were also quantified. While no substantial differences in the speed of locomotion were found, the directionality of cell movement was greatly promoted by the presence of calreticulin. Calreticulin expression was also found to have a dramatic effect on the phosphorylation state of serine 636 of IRS-1, such that phosphorylation of IRS-1 on serine 636 increased radically in the absence of calreticulin. Most importantly, treatment of cells with the RhoA/ROCK inhibitor, Y-27632, which among its many effects also inhibited serine 636 phosphorylation of IRS-1, had profound effects on cell-substratum adhesion, in that it suppressed focal contacts, induced extensive close contacts, and increased the strength of adhesion. The latter effect, while counterintuitive, can be explained by the close contacts comprising labile bonds but in large numbers. In addition, the lability of bonds in close contacts would permit fast locomotion. An interesting and novel finding is that Y-27632 treatment of MEFs releases them from contact inhibition of locomotion, as evidenced by the invasion of a cell's underside by the thin lamellae and filopodia of a cell in close apposition.

Calreticulin induces dilated cardiomyopathy.

BACKGROUND: Calreticulin, a Ca(2+)-buffering chaperone of the endoplasmic reticulum, is highly expressed in the embryonic heart and is essential for cardiac development. After birth, the calreticulin gene is sharply down regulated in the heart, and thus, adult hearts have negligible levels of calreticulin. In this study we tested the role of calreticulin in the adult heart. METHODOLOGY/PRINCIPAL FINDINGS: We generated an inducible transgenic mouse in which calreticulin is targeted to the cardiac tissue using a Cre/loxP system and can be up-regulated in adult hearts. Echocardiography analysis of hearts from transgenic mice expressing calreticulin revealed impaired left ventricular systolic and diastolic function and impaired mitral valve function. There was altered expression of Ca(2+) signaling molecules and the gap junction proteins, Connexin 43 and 45. Sarcoplasmic reticulum associated Ca(2+)-handling proteins (including the cardiac ryanodine receptor, sarco/endoplasmic reticulum Ca(2+)-ATPase, and cardiac calsequestrin) were down-regulated in the transgenic hearts with increased expression of calreticulin. CONCLUSIONS/SIGNIFICANCE: We show that in adult heart, up-regulated expression of calreticulin induces cardiomyopathy in vivo leading to heart failure. This is due to an alternation in changes in a subset of Ca(2+) handling genes, gap junction components and left ventricle remodeling.

Evidence for calreticulin attenuation of cardiac hypertrophy induced by pressure overload and soluble agonists.

While calreticulin has been shown to be critical for cardiac development, its role in cardiac pathology is unclear. Previous studies have shown the detrimental effects on the heart of sustained germline calreticulin overexpression, yet without calreticulin, the heart does not develop normally. Thus, carefully balanced calreticulin levels are required for the heart to develop and to function properly into adulthood. But what happens to calreticulin levels, and how is this regulated, during cardiac hypertrophy, during which the fetal gene program is reactivated, at least partially? Our working hypothesis was that c-Src, a kinase whose activity we previously found to be correlated with calreticulin expression, was involved with calreticulin in regulating the response to hypertrophic signals. Thus, we subjected adult mice to transverse aortic constriction to induce left ventricular hypertrophy. We found that aortic constriction caused calreticulin levels to increase, whereas those of c-Src fell with longer constriction time. We also examined the ability of embryonic stem cell-derived cardiomyocytes to respond to soluble hypertrophic agonists. Endothelin-1 treatment caused a significantly greater cell area increase of calreticulin-null cardiomyocytes, which had higher c-Src activity, compared with wild-type cells. c-Src inhibition abolished this difference. Greater c-Src activity may explain the efficacy with which calreticulin-null cells are able to induce the hypertrophic program, while cells containing calreticulin may be able to attenuate the hypertrophic response as a result of decreased c-Src activity. Thus, calreticulin may have a protective effect on the heart in the face of cardiac hypertrophy.

Calreticulin and focal-contact-dependent adhesion.

Cell adhesion is regulated by a variety of Ca2+-regulated pathways that depend on Ca2+-binding proteins. One such protein is calreticulin, an ER-resident protein. Calreticulin signalling from within the ER can affect processes outside the ER, such as expression of several adhesion-related genes, most notably vinculin and fibronectin. In addition, changes in the expression level of calreticulin strongly affect tyrosine phosphorylation of cellular proteins, which is known to affect many adhesion-related functions. While calreticulin has been localized to cellular compartments other than the ER, it appears that only the ER-resident calreticulin affects focal-contact-dependent adhesion. In contrast, calreticulin residing outside the ER may be involved in contact disassembly and other adhesion phenomena. Here, we review the role of calreticulin in focal contact initiation, stabilization, and turnover. We propose that calreticulin may regulate cell-substratum adhesion by participating in an "ER-to-nucleus" signalling and in parallel "ER-to-cell surface" signalling based on posttranslational events.

Embryonic stem cell-derived cardiomyogenesis: a novel role for calreticulin as a regulator.

A role for calreticulin, an endoplasmic reticulum (ER)-resident, Ca(2+)-binding chaperone, has recently emerged in the context of cardiomyogenesis. We previously proposed calreticulin to be a novel cardiac fetal gene, because calreticulin knockout causes embryonic lethality in mice as a result of cardiac defects, it is transiently activated during heart development, and heart-targeted overexpression of constitutively active calcineurin in calreticulin-null mice rescues the lethal phenotype. Calreticulin affects Ca(2+) homeostasis and expression of adhesion-related genes. Using cardiomyocytes derived from both calreticulin-null and wild-type embryonic stem (ES) cells, we show here that cardiomyogenesis from calreticulin-null ES cells is accelerated but deregulated, such that the myofibrils of calreticulin-null cardiomyocytes become disorganized and disintegrate with time in culture. We have previously shown that the disorganization of the actin cytoskeleton in calreticulin-null cells may be explained, at least in part, by the downregulation of adhesion proteins, implying that calreticulin ablation causes adhesion-related defects. Here, upon examination of adhesion proteins, we found that vinculin is downregulated in calreticulin-null cardiomyocytes. We also found c-Src activity to be higher in calreticulin-null cardiomyocytes than in wild-type cardiomyocytes, and c-Src activity is affected by both calreticulin and [Ca(2+)]. Finally, we show that calreticulin and calsequestrin, the major Ca(2+) storage proteins of the ER and sarcoplasmic reticulum, respectively, exhibit alternate distributions. This suggests that calreticulin may have a housekeeping role to play in mature cardiomyocytes as well as during cardiomyogenesis. We propose here that calreticulin, an ER Ca(2+) storage protein, is a crucial regulator of cardiomyogenesis whose presence is required for controlled cardiomyocyte development from ES cells.

Expression of endoplasmic reticulum chaperones in cardiac development.

To determine if cardiogenesis causes endoplasmic reticulum stress, we examined chaperone expression. Many cardiac pathologies cause activation of the fetal gene program, and we asked the reverse: could activation of the fetal gene program during development induce endoplasmic reticulum stress/chaperones? We found stress related chaperones were more abundant in embryonic compared to adult hearts, indicating endoplasmic reticulum stress during normal cardiac development. To determine the degree of stress, we investigated endoplasmic reticulum stress pathways during cardiogenesis. We detected higher levels of ATF6alpha, caspase 7 and 12 in adult hearts. Thus, during embryonic development, there is large protein synthetic load but there is no endoplasmic reticulum stress. In adult hearts, chaperones are less abundant but there are increased levels of ATF6alpha and ER stress-activated caspases. Thus, protein synthesis during embryonic development does not seem to be as intense a stress as is required for apoptosis that is found during postnatal remodelling.

Kinase-dependent adhesion to fibronectin: regulation by calreticulin.

We studied the phosphorylation (activation status) of c-Src and CaMKII in MEFs either wild type for calreticulin, calreticulin-null, or rescued with full-length calreticulin. We found that calreticulin-null cells were poorly spread on the substratum and formed few, if any, focal contacts. Fibronectin expression and deposition were lower in calreticulin-null MEFs compared to calreticulin-expressing cells, which also exhibited increased c-Src and CaMKII phosphorylation (activity). Plating MEFs on preformed fibronectin rescued the poor adhesive phenotype of calreticulin-null cells, and caused a decrease in c-Src Y418 phosphorylation (activity). c-Src inhibition caused the calreticulin-null MEFs to become well spread on the substratum and to make many prominent focal contacts. Calmodulin and CaMKII inhibition caused similar results, along with a notable increase in paxillin phosphorylation (activation). To test if the calcium storage function of calreticulin was responsible for these effects, we manipulated intracellular [Ca(2+)]. Lowering [Ca(2+)](ER) caused an increase in c-Src phosphorylation and a decrease in fibronectin abundance. Conversely, increasing [Ca(2+)] caused opposite effects. These results suggest that calreticulin regulates both the c-Src and calmodulin/CaMKII pathways, enabling cells to be better spread on the substratum by allowing greater fibronectin deposition and increased focal contact formation.

Analysis of the suitability of calreticulin inducible HEK cells for adhesion studies: microscopical and biochemical comparisons.

Calreticulin is a Ca(2+)-buffering ER chaperone that also modulates cell adhesiveness. In order to study the effect of calreticulin on the expression of adhesion-related genes, we created a calreticulin inducible Human Embryonic Kidney (HEK) 293 cell line. We found that fibronectin mRNA and both intra- and extra-cellular fibronectin protein levels increased following calreticulin induction. However, despite this increase in fibronectin, HEK293 cells did not assemble an extracellular fibrillar fibronectin matrix regardless of the level of calreticulin expression. Furthermore, HEK293 cells exhibited a poorly organized actin cytoskeleton, did not have clustered fibronectin receptors at the cell surface, and did not form focal contacts. This likely accounts for the lack of fibronectin matrix deposition by these cells regardless of calreticulin expression level. Vinculin abundance did not appreciably increase upon calreticulin induction and the level of active c-Src, a regulatory kinase of focal contacts, was found to be abundant and unregulated by calreticulin induction in these cells. The inability to form stable focal contacts and to commence fibronectin fibrillogenesis due to high c-Src activity may be responsible for the poor adhesive phenotype of HEK 293 cells. Thus, we show here that HEK293 cells are not suitable for microscopical studies of cell-substratum adhesions, but are best suited for biochemical studies.

Differential calreticulin expression affects focal contacts via the calmodulin/CaMK II pathway.

Calreticulin is an ER calcium-storage protein, which influences gene expression and cell adhesion. In this study, we analysed the differences in adhesive properties of calreticulin under- and overexpressing fibroblasts in relation to the calmodulin- and calcium/calmodulin-dependent kinase II (CaMK II)-dependent signalling pathways. Cells stably underexpressing calreticulin had elevated expression of calmodulin, activated CaMK II, activated ERK and activated c-src. Inhibition of calmodulin by W7, and CaMK II by KN-62, caused the otherwise weekly adhesive calreticulin underexpressing cells to behave like the overexpressing cells, via induction of increased cell spreading. Increased vinculin, activated paxillin, activated focal adhesion kinase and fibronectin levels were observed upon inhibition of either the calmodulin or the CaMK II signalling pathways, which was accompanied by an increase in cell spreading and focal contact formation. Both KN-62 and W7 treatment increased cell motility in underexpressing cells, but W7 treatment led to loss of directionality. Thus, both the calmodulin and CaMK II signalling pathways influence cellular spreading and motility, but subtle differences exist in their distal effects on motility effectors.

Calreticulin affects fibronectin-based cell-substratum adhesion via the regulation of c-Src activity.

Calreticulin is an endoplasmic reticulum Ca2+-storage protein, which influences gene expression and cell adhesion. In this study, we show that calreticulin induces fibronectin gene expression and matrix deposition, leading to differences in cell spreading and focal adhesion formation in cells differentially expressing calreticulin. We further show that these effects of calreticulin occur via a c-Src-regulated pathway and that c-Src activity is inversely related to calreticulin abundance. Since c-Src is an important regulator of focal contact turnover, we investigated the effect of c-Src inhibition on cells differentially expressing calreticulin. Inhibition of c-Src rescued the poorly adhesive phenotype of the calreticulin-underexpressing cells in that they became well spread, commenced formation of numerous focal contacts, and deposited a rich fibronectin matrix. Importantly, we show that c-Src activity is dependent on releasable Ca2+ from the endoplasmic reticulum, thus implicating Ca2+-sensitive pathways that are affected by calreticulin in cell-substratum adhesion. We propose that calreticulin affects fibronectin synthesis and matrix assembly via the regulation of fibronectin gene expression. In parallel, calcium-dependent effects of calreticulin on c-Src activity influence the formation and/or stability of focal contacts, which are instrumental in matrix assembly and remodeling.

Dissecting focal adhesions in cells differentially expressing calreticulin: a microscopy study.

BACKGROUND INFORMATION: Our previous studies have shown that calreticulin, a Ca2+-binding chaperone located in the endoplasmic reticulum, affects cell-substratum adhesions via the induction of vinculin and N-cadherin. Cells overexpressing calreticulin contain more vinculin than low expressers and make abundant contacts with the substratum. However, cells that express low levels of calreticulin exhibit a weak adhesive phenotype and make few, if any, focal adhesions. To date, the identity of the types of focal adhesions made by calreticulin overexpressing and low expressing cells has not been dissected. RESULTS: The results of the present study show that calreticulin affects fibronectin matrix assembly in L fibroblast cell lines that differentially express the protein, and that these cells also differ profoundly in focal adhesion formation. Although the calreticulin overexpressing cells generate numerous interference-reflection-microscopy-dark, vinculin- and paxillin-containing classical focal contacts, as well as some fibrillar adhesions, the cells expressing low levels of calreticulin generate only a few weak focal adhesions. The fibronectin receptor was found to be clustered in calreticulin overexpressing cells, but diffusely distributed over the cell surface in low expressing cells. Plating L fibroblasts on fibronectin-coated substrata induced extensive spreading in all cell lines tested. However, although calreticulin overexpressing cells were induced to form classical vinculin-rich focal contacts, the low calreticulin expressing cells overcame their weak adhesive phenotype by induction of many tensin-rich fibrillar adhesions, thus compensating for the low level of vinculin in these cells. CONCLUSIONS: We propose that calreticulin affects fibronectin production and, thereby, assembly, and it indirectly influences the formation and/or stability of focal contacts and fibrillar adhesions, both of which are instrumental in matrix assembly and remodelling.

Ultrastructural analysis of development of myocardium in calreticulin-deficient mice.

BACKGROUND: Calreticulin is a Ca2+ binding chaperone of the endoplasmic reticulum which influences gene expression and cell adhesion. The levels of both vinculin and N-cadherin are induced by calreticulin expression, which play important roles in cell adhesiveness. Cardiac development is strictly dependent upon the ability of cells to adhere to their substratum and to communicate with their neighbours. RESULTS: We show here that the levels of N-cadherin are downregulated in calreticulin-deficient mouse embryonic hearts, which may lead to the disarray and wavy appearance of myofibrils in these mice, which we detected at all investigated stages of cardiac development. Calreticulin wild type mice exhibited straight, thick and abundant myofibrils, which were in stark contrast to the thin, less numerous, disorganized myofibrils of the calreticulin-deficient hearts. Interestingly, these major differences were only detected in the developing ventricles while the atria of both calreticulin phenotypes were similar in appearance at all developmental stages. Glycogen also accumulated in the ventricles of calreticulin-deficient mice, indicating an abnormality in cardiomyocyte metabolism. CONCLUSION: Calreticulin is temporarily expressed during heart development where it is required for proper myofibrillogenesis. We postulate that calreticulin be considered as a novel cardiac fetal gene.

Identification of an N-domain histidine essential for chaperone function in calreticulin.

Calreticulin is an endoplasmic reticulum (ER) luminal Ca(2+)-binding chaperone involved in folding of newly synthesized glycoproteins via the "calreticulin-calnexin cycle." We reconstituted ER of calreticulin-deficient cells with N-terminal histidine (His25, His82, His128, and His153) calreticulin mutants and carried out a functional analysis. In crt(-/-) cells bradykinin-dependent Ca2+ release is altered, and the reestablishment of bradykinin-dependent Ca2+ release was used as a marker for calreticulin function. Bradykinin-dependent Ca2+ release from the ER was rescued by wild type calreticulin and by the His25, His82, or His128 mutant but not by the His153 mutant. Wild type calreticulin and the His25, His82, and His128 mutants all prevented in vitro thermal aggregation of malate dehydrogenase and IgY, whereas the His153 mutant did not, indicating that His153 chaperone function was impaired. Biophysical analysis of His153 mutant revealed that conformation changes in calreticulin mutant may be responsible for the loss of its chaperone activity. We conclude that mutation of a single amino acid residue in calreticulin has devastating consequences for its chaperone function, indicating that mutations in chaperones may play a significant role in protein folding disorders.