Protein Kinase D1 regulates MMP expression and inhibits breast cancer cell invasion

Breast Cancer Research 2009, 11:R13doi:10.1186/bcr2232

Tim Eiseler , Heike Doeppler , Irene K Yan , Steve Goodison and Peter Storz

We found that the serine/threonine kinase Protein Kinase D1(PKD1) is highly expressed in ductal epithelial cells of normal human breast tissue, but is reduced in its expression in >95% of all analyzed samples of human invasive breast tumors. Additionally, PKD1 is not expressed in highly invasive breast cancer cell lines, whereas non- or very low-invasive breast cancer cell lines express PKD1. Our results further implicate that in MDA-MB-231 cells PKD1 expression is blocked by epigenetic silencing via DNA methylation. The re-expression of constitutively-active PKD1 in MDA-MB-231 cells drastically reduced their ability to invade in 2D and 3D cell culture. Moreover, MCF-7 cells acquired the ability to invade in 2D and 3D cell culture when PKD1 expression was knocked-down by shRNA. PKD1 also regulated the expression of breast cancer cell matrix-metalloproteinases MMP-2, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, MMP-14 and MMP-15, providing a potential mechanism for PKD1 mediation of the invasive phenotype.

The spliceosome as target for anticancer treatment
van Alphen RJ, Wiemer EA, Burger H, Eskens FA. “The spliceosome as target for anticancer treatment”, Br J Cancer. 2009 Jan 27;100(2):228-32


Es una revisión interesante donde se ven aspectos básicos del “spliceosome”.
Sin embargo hay algunas cuestiones que me inquietan: 1) ¿En general los niveles de RNAm o proteina de hnRSPs y SRs están modificados en el cancer? Algo relacionado a esta pregunta que si se conoce es que en algunos genes como BRCA1, p53, MSH2, etc, tienen mutaciones en los “splice site” y algunas de estas son consideradas SNPs. Por otro lado un grupo chino ha correlacionado con tamaño de adenocarcinoma de pulmon con la presencia de hnRNP K. Además este grupo ha demostrado que un siRNA dirigido para hnRNP k inhibe el crecimiento de la línea celular de adenocarcinoma A549. Aunque este dato podría interpretarse como que hnRNP k está involucrado en este tipo de cáncer, también podría deberse a que al quitar este elemento del spliceosma afecte a genes fundamentales para la célula sin importar que esta sea maligna. Como un comentario, quiero resaltar que estos artículos están en chino¡¡¡ (porque están escritos en chino¡¡¡).

ii) Si están aumentados o disminuidos en el cáncer, ¿Cuáles podrían ser los elementos que regulan su expresión? En primera instancia se me ocurre un análisis de las secuencias promotoras de las hnRNP o SR que cambien su expresión. Sin embargo, algo que se me ocurrió y que ya se describió es que estas moléculas podrían auto-regularse. Al menos para hnRNP L existe un mecanismo de tipo feedback negativo. En tal artículo, ellos demuestran que hnRNP-L induce la incorporación de un codón de termino prematuro contenido en el exón 6 produciendo el NMD (nonsense-mediated decay) (http://mcb.asm.org/cgi/reprint/MCB.01689-08v1?view=long&pmid=19124611). Aunque tambien se deberian tomar encuenta otros procesos como los miRNAs.

iii) Si la premisa de que las hnRNPs y SR están involucradas en el cáncer, me imagino que habrá algunas de estas moléculas que funcionen como grandes reguladores de ellas mismas y del splacing alternativo por lo que el desarrollo de pequeñas moléculas dirigidas contra ellas será una opción. La pregunta que me hago relacionada con esta idea es ¿Cómo le podría hacer para descubrir tales reguladores? Una opción sería investigar cuales son los genes que en el cáncer presentan más de una isoforma generada por el splacing alternativo cuando se comparan contra las células normales y posteriormente determinar cuales son los elementos “splice site” que comparten y posteriormente identificar cuáles son las proteínas que se unen a tales sitios, esperando que algunas sean hnRNPs.


Por último, en esta revisión se propone tres estrategias para modular este mecanismo con el objetivo de obtener una actividad antitumoral:
i) Splice site modulation
ii) Targeting of variant proteins
iii) Spliceosoma inhibitors

Un Déjà vu: la seguridad de la terapia génica y el tratamiento con células troncales en la clínica.

Donor-Derived Brain Tumor Following Neural Stem Cell Transplantation in an Ataxia Telangiectasia Patient

Ninette Amariglio^1,2, Abraham Hirshberg³, Bernd W. Scheithauer⁴, Yoram Cohen¹, Ron Loewenthal⁵, Luba Trakhtenbrot², Nurit Paz¹, Maya Koren-Michowitz², Dalia Waldman⁶, Leonor Leider-Trejo⁷, Amos Toren⁶, Shlomi Constantini⁸, Gideon Rechavi^1,6*

Background

Neural stem cells are currently being investigated as potential therapies for neurodegenerative diseases, stroke, and trauma. However, concerns have been raised over the safety of this experimental therapeutic approach, including, for example, whether there is the potential for tumors to develop from transplanted stem cells.

Methods and Findings

A boy with ataxia telangiectasia (AT) was treated with intracerebellar and intrathecal injection of human fetal neural stem cells. Four years after the first treatment he was diagnosed with a multifocal brain tumor. The biopsied tumor was diagnosed as a glioneuronal neoplasm. We compared the tumor cells and the patient's peripheral blood cells by fluorescent in situ hybridization using X and Y chromosome probes, by PCR for the amelogenin gene X- and Y-specific alleles, by MassArray for the ATM patient specific mutation and for several SNPs, by PCR for polymorphic microsatellites, and by human leukocyte antigen (HLA) typing. Molecular and cytogenetic studies showed that the tumor was of nonhost origin suggesting it was derived from the transplanted neural stem cells. Microsatellite and HLA analysis demonstrated that the tumor is derived from at least two donors.

Conclusions

This is the first report of a human brain tumor complicating neural stem cell therapy. The findings here suggest that neuronal stem/progenitor cells may be involved in gliomagenesis and provide the first example of a donor-derived brain tumor. Further work is urgently needed to assess the safety of these therapies.

http://medicine.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pmed.1000029#toclink1

Muerte celular

Les anexo el artículo con las recomendaciones del comité de nomenclatura de muerte celular.

2009, Classification of cell death.pdf

Entosis: cell death by invasion

News and Views

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Nature Cell Biology 9, 1346 (2007)
doi:10.1038/ncb1207-1346

Most animal cells require attachment to an extracellular matrix for survival. Within the mammary gland, death of matrix-deprived cells underlies lumen formation during development and is also thought to act as a defence against the formation of breast tumours. It has long been known that epithelial cells detached from their extracellular matrix can undergo a process of apoptotic cell death called anoikis. Overholtzer et al.(Cell10.1016/j.cell.2007.10.040; 2007) now report that detached epithelial cells can also take an alternative path to death which is driven by cell-in-cell invasion.

While observing mammary epithelial cells in suspension cultures, they found that these detached cells are often internalized into vacuoles within neighbouring cells. Following their internalization, most of these cells are eventually degraded by lysosomal enzymes; however, some of the captured cells are instead released. Surprisingly, a small percentage of cells may undergo cell division while entrapped within other cells.

Internalization is independent of apoptosis and instead occurs by a process the authors have named entosis, from the Greek entos, meaning inside or within. The study showed that live internalizing cells actively participate in the invasion process, in a manner dependent on signalling through the small GTPase Rho, which mediates actomyosin-mediated contraction. Overholtzer et al. also noticed that before internalization, cells establish E-cadherin–beta-catenin-mediated adherens junctions and the compaction of these junctions appears to directly mediate the entosis process. The authors propose that invasion is driven by unbalanced myosin II-dependent forces during compaction.

Is entosis an artefact of cells cultured in suspension or is it a physiologically relevant phenomenon that has thus far escaped the attention of cell biologists? To address this question, the authors obtained pleural fluid from breast metastatic tumours as well as solid primary breast tumour samples, as the features exhibited by internalized cells are reminiscent of 'cell-in-cell' or cell 'cannibalism' images observed in metastatic cells. They discovered that cells from both classes of tumours exhibit characteristics of entosis: cell-in-cell intermediates with high levels of adherent junction proteins were observed as well as cells inside the lysosomes of other cells.

The study by Overholtzer et al. suggests that a cytological feature known to clinicians for many years may be crucial for the growth of a wide variety of tumours, possibly as a tumour-suppressor mechanism that eliminates cells that have escaped their natural environment and are detached from the normal matrix. There is definitely more to entosis than just another way to die.

Nuevo metodo util para varios de nuestros protocolos

Novel egg white–based 3-D cell culture system
Although three dimensional (3-D) cell culture systems have numerous advantages over traditional monolayer culture, the currently available
3-D cell culture media are cost-prohibitive for regular use by the majority of research laboratories. Here we show a simple system
based on avian egg white that supports growth of cells in 3-D, at a significantly decreased cost. Specifically, we show that growth of immortalized
human breast epithelial cells (MCF10A) in egg white–based medium results in formation of acini with hollow lumens, apoptotic
clearance of the cells in the lumen, and apicobasal polarization comparable to what has been described using established 3-D culture
media such as reconstituted basement membrane preparations (BM). There was no significant difference in MCF10A proliferation and
acinar size between egg white and BM. We also cultured different established cell lines, oncogene-transformed MCF10A, and mouse mammary
epithelial cells in egg white and BM, and observed similar morphology. In summary, our data convincingly argue that egg white can
be used as a suitable alternative model for 3-D cell culture studies. We strongly believe that this simple and inexpensive method should allow
researchers to perform 3-D cell culture experiments on a regular basis, and result in a dramatic increase of use of the 3-D cell culture
in research. Thus, this finding lays the foundation for significantly increased, cost-effective use of 3-D cultures in cell biology.
Art disponible en https://cancerfunctionalgenomic.wikispaces.com/Tecnicas

Caspase-8

Cancer Res. 2008 Jun 15;68(12):4491-3.
Links
Caspase-8: fly or die.
Frisch SM.
West Virginia University, Mary Babb Randolph Cancer Center, Morgantown, West Virginia, USA. sfrisch@hsc.wvu.edu
Recent studies have revealed that procaspase-8 has an important function in cell adhesion and motility. Src phosphorylation controls this function by preventing the conversion of procaspase-8, which is an adhesion/migration factor, to mature caspase-8, which is an apoptosis-inducing factor. This provides a mechanism to switch these opposing functions. In its migratory role, procaspase-8 interacts with the phosphatidylinositol-3-OH kinase regulatory subunit p85alpha and c-src to modulate signaling by Rac and extracellular signal-regulated kinase, and promote calpain activation. Here, I survey the findings of these studies and discuss potential mechanisms and ramifications for cancer prognosis and therapy.
PMID: 18559490 [PubMed - in process]

Cancer Res. 2007 Dec 15;67(24):11505-9.
Links
Caspase-8 interacts with the p85 subunit of phosphatidylinositol 3-kinase to regulate cell adhesion and motility.
Senft J, Helfer B, Frisch SM.
Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, West Virginia 26506, USA.
Cell migration plays an important role in tumor cell invasion and metastasis. Previously, we reported that caspase-8 contributes to cell migration and adhesion, a novel nonapoptotic function of an established apoptotic factor. Herein, we report that pro-caspase-8 is capable of restoring cell migration/adhesion to caspase-8-null cells, establishing the first biological function of a pro-caspase. The catalytic activity of caspase-8 was not required for cell motility. Stimulation of motility with epidermal growth factor induced the phosphorylation of caspase-8 on tyrosine-380 and the interaction of caspase-8 with the p85 alpha subunit of phosphatidylinositol 3-kinase. Tyrosine-380 was required for the restoration of cell motility and cell adhesion in caspase-8-null cells, demonstrating the importance of the caspase-8-p85 interaction for these nonapoptotic functions. These results suggest that caspase-8 phosphorylation converts it from a proapoptotic factor to a cell motility factor that, through tyrosine-380, interacts with p85, an established cell migration component.
PMID: 18089778 [PubMed - indexed for MEDLINE]

Una cosa por otra: H. pilory protects children from asthma

H. pilory es una bacteria que en humanos puede causar gastritis y úlceras, sin embargo, recientemente se ha encontrado que tanto en adultos como en niños las presencia de H. pilory disminuye el riesgo de asma. Esto nos lleva a pensar que al erradicar esta bacteria estamos dejando más susceptible a la población humana de desarrollar asma

"Among teens and children ages 3 to 19 years, carriers of H. pylori were 25 percent less likely to have asthma."

The impact was even more potent among children ages 3 to 13: they were 59 percent less likely to have asthma if they carried the bacterium, the researchers report. H. pylori carriers in teens and children were also 40 percent less likely to have hay fever and associated allergies such as eczema or rash."

"These results, which follow on from similar findings in adults published by the same authors last year, are based on an analysis of data gathered from 7,412 participants in the fourth National Health and Nutrition Survey (NHANES IV) conducted from 1999 to 2000 by the National Center for Health Statistics"

"Asthma has been rising steadily for the past half-century. Meanwhile H. pylori, once nearly universal in humans, has been slowly disappearing from developed countries over the past century due to increased antibiotic use, which kills off the bacteria, and cleaner water and homes"

http://www.sciencedaily.com/releases/2008/07/080715071419.htm

Cell lineage analysis of a mouse tumor.

1: Cancer Res. 2008 Jul 15;68(14):5924-31.

Cell lineage analysis of a mouse tumor.

Frumkin D, Wasserstrom A, Itzkovitz S, Stern T, Harmelin A, Eilam R, Rechavi G, Shapiro E.

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.

Revealing the lineage relations among cancer cells can shed light on tumor growth patterns and metastasis formation, yet cell lineages have been difficult to come by in the absence of a suitable method. We previously developed a method for reconstructing cell lineage trees from genomic variability caused by somatic mutations. Here, we apply the method to cancer and reconstruct, for the first time, a lineage tree of neoplastic and adjacent normal cells obtained by laser microdissection from tissue sections of a mouse lymphoma. Analysis of the reconstructed tree reveals that the tumor initiated from a single founder cell, approximately 5 months before diagnosis, that the tumor grew in a physically coherent manner, and that the average number of cell divisions accumulated in cancerous cells was almost twice than in adjacent normal lung epithelial cells but slightly less than the expected figure for normal B lymphocytes. The cells were also genotyped at the TP53 locus, and neoplastic cells were found to share a common mutation, which was most likely present in a heterozygous state. Our work shows that the ability to obtain data regarding the physical appearance, precise anatomic position, genotypic profile, and lineage position of single cells may be useful for investigating cancer development, progression, and interaction with the microenvironment.

Human Breast cancer cells traced to transplanted stem cells on humans

Human Breast cancer cells traced to transplanted stem cells on humans

Martha Kerr Reuters Health Last Modified: June 26, 2008

Last Updated: 2008-06-26 16:24:49 -0400 (Reuters Health) NEW YORK (Reuters Health) - Stem cells can give rise to both breast cancer cells and the cells of the tumor microenvironment. Furthermore, stem cells transplanted from a donor can be the source of breast cancer in a transplant recipient. "This is a whole new way of thinking about stem cells and cancer," Dr. Sanford H. Barsky told Reuters Health about research he is presenting at the Department of Defense's Era of Hope Breast Cancer Research meeting that has just convened in Baltimore, Maryland. Dr. Barsky, of The Ohio State University in Columbus, and colleagues conducted two phases of study. One phase was a search of a bone marrow and organ transplant registry, in which "we noticed the origins of some cancers were from the donor organ," Dr. Barsky said. "Cancer-promoting donor stem cells lodged in different sites in the recipients and developed." In the second phase of their research, conducted in mice, the Ohio team tagged bone marrow cells before transplantation. The researchers were able to observe that "breast cancer doesn't just arise in breast tissue and everything that contributes to the breast cancer microenvironment doesn't reside only in the breast." "Invasive breast cancers reflect the presence of both cancer-promoting as well as cancer-initiating stem cells derived from ectopic locations," Dr. Barsky explained. "It appears that breast cancers and the tumors' microenvironment both arise from stem cells." "Inflammatory breast cancers form from emboli in vascular channels, and these channels form the microenvironment. Both arise from donor stem cells and there is cross-talk between them," the researcher added. In the abstract for their presentation, the investigators write, "These emboli, which were resistant to chemotherapy, exhibited a prominent stem cell-like phenotype...suggesting that the lymphovascular tumor emboli, like the human embryonal blastocyst, are derived from stem cells locked in self-renewal... some of the endothelial cells which lined the channels containing the tumor emboli exhibited evidence of bone marrow origin." "This shows that we may be able to turn off cancer cells at the level of the stem cell," Dr. Barsky said. "If we can't stop the stem cells, then maybe we could replace the cancer cells with normal cells." "Breast cancer is really a rare disease at the cellular level, despite the fact that it is fairly common on a population basis...and stem cells are rare in the breast," Dr. Barsky noted. "Our research could lead to a way of tagging cancer cells from their point of origin and may provide a new method of drug delivery." http://www.oncolink.com/resources/article.cfm?c=3&s=8&ss=23&Year=2008&Month=06&id=15400

Is Tumor Growth Sustained by Rare Cancer Stem Cells or Dominant Clones?

les recomiendo que lean este artículo de revisión de Cancer Research:

A key issue for cancer biology and therapy is whether the relentless growth of a tumor is driven by a substantial proportion of its cells or exclusively by a rare subpopulation, commonly termed "cancer stem cells." Support for the cancer stem cell model has been stimulated by experiments in which human tumor cells were transplanted into immunodeficient mice. Most notably, in human acute myeloid leukemia, only a minute proportion of the cells, displaying a defined phenotype, could seed leukemia in mice. Xenotransplantation, however, may fail to reveal many tumor growth–sustaining cells because the foreign microenvironment precludes essential interactions with support cells. In studies that instead have transplanted mouse leukemias and lymphomas into syngeneic animals, most of the tumors seem to be maintained by the dominant cell population, and only a few types of mouse leukemia seem to be sustained by a minor tumor growth–sustaining subpopulation. The collective evidence suggests that various tumors may span the spectrum between the extremes represented by the two models. If tumor growth can indeed be sustained either by rare cancer stem cells or dominant clones or both, as current evidence suggests, curative therapy for many types of tumors will most likely require targeting all the tumor cell populations. [Cancer Res 2008;68(11):4018–21]

http://canreviews.aacrjournals.org/cgi/reprint/canres;68/11/4018

Nuevo mecanismo de regulacion postraduccional

[Protein Synthesis, Post-Translational Modification, and Degradation] Adenosine Receptors Control a New Pathway of Fas-associated Death Domain Protein Expression Regulation by Secretion
from Journal of Biological Chemistry current issue by Tourneur, L., Mistou, S., Schmitt, A., Chiocchia, G.

FADD is the key adaptor transmitting the apoptotic signal mediated by death receptors. We have previously shown that FADD protein expression could be lost in vivo in cancerous cells, in mice and humans, and be used as prognostic factor. Furthermore, loss of FADD could contribute to tumor progression and aggressiveness. However, the mechanism accounting for the loss of FADD was unknown. Using in vitro-cultured mouse organ models, we demonstrated that loss of FADD occurred through a new regulatory pathway of FADD expression by secretion. The secretion of FADD is an active release following shedding of microvesicles derived from the plasma membrane. In our experimental settings, this phenomenon was restricted to 6 of 12 FADD-expressing organs. This process is calcium- and adenosine-dependent. Moreover, we identified the two receptors with low affinity to adenosine, namely A2B and A3 adenosine receptors, as regulators of the FADD secretion process. Furthermore, we showed that modulating A3 adenosine receptor can convert a nonsecreting organ into a FADD-secreting one. Finally, we reported that mouse FADD release occurred in vivo during tumor disease. These results demonstrate the existence of a new localization site (in microvesicles) and regulatory mechanism (by secretion) of the FADD protein, and the implication of adenosine receptors in this process. These data open a new field of investigation consisting of the possibility to regulate FADD expression via the modulation of adenosine receptors, which constitutes a therapeutic target in diseases in which FADD-mediated signaling is impaired.