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Intracellular Transport and Tumorigenesis

alexandre mironov

[IFOM]

Alexandre A. Mironov, MD, PhD, DSc (Dr. habil. )
c/o IFOM-IEO Campus
Via Adamello, 16 - 20139 Milan, Italy
Tel. +39 02 574303869 - Fax. +39 02 574303231
alexandre.mironovmailifom.eu

Research projects

fig 1fig.1, The three-dimensional view of the Golgi apparatus after tomographic reconstruction.
[+ zoom]

The group studies models of intracellular transport and some aspects of tumorigenesis related to intracellular transport. Membrane and secretory proteins and membrane lipids are synthesized in the endoplasmic reticulum (ER). After their synthesis, folding and quality control, these proteins exit from the ER and are then transported along the secretory pathway to the Golgi apparatus (GA). The GA occupies a central position within the classical secretory pathway and in multiple recycling routes. In spite of an accumulation of a huge amount of information about the GA and protein machines operating there, mechanisms of intra-Golgi transport remain obscure and unresolved.

Alexandre A. Mironov has long worked on the mechanisms of intracellular transport managing to refute some of the pre-existing dogmas; in particular, that COPII vesicles are not carriers of proteins, but serve to regulate the transport through the Golgi apparatus. He found that during the transport through the GA, narrow tubes interconnect Golgi cisternae. He has described transporting and non-transporting Golgi stacks (Fig. 1 and 2) and proposed and justified a new model of intracellular transport, namely, the kiss-and-run model.

The group uses all modern methods of microscopy, in particular he is a leader in the development and application of correlative light and electron microscopy, tomography and immuno cryomicroscopy.

The main direction is the development of early and specific clinical cancers tests.

Exosome and the new role of double stranded RNA for cancer and evolution. This project will justify the method of diagnosis of early stages of cancer regardless of its location and origin, including the measurement of the RNA content in blood plasma that is different from other methods in such a way that in order to improve diagnostic accuracy it is necessary to measure a concentration of not single chains of RNA or small RNAs, but the total content of double stranded RNA (dsRNA) of 18-25 nucleotides in length. If a significant increase in the concentration of dsRNA were discovered there, an additional ultrasound or another type of specific tests of cancer should be performed.

fig 2fig.2, Structure of the Golgi apparatus in cells where exit of cargo from the endoplasmic reticulum is blocked. There are a lot of COPI vesicles. [+ zoom]

The hypothesis poses that miRNA that were detected in blood of patients suffering from cancer are double stranded RNAs and appearance of miRNA is a result of synonymous or non-synonymous substitution of nucleotide in coding regions of DNA. In contrast, to the current consensus, it has been proposed that these miRNAs are short double-stranded RNA (dsRNA) that are formed after synonymous mutations leading to intra-molecular (formation of hairpins) or inter-molecular (gluing of two RNAs) hybridization of pre-mRNA and mRNAs that are synthesized on the mutated regions of DNA and that formation of a tumour is accompanied by the secretion into the blood of double stranded RNAs (dsRNAs) composed 19-25 nucleotides in length. Secretion of these dsRNAs is the result of accumulation of synonymous and non-synonymous substitutions in pre-cancer cells that initially do not have any phenotypic effect but lead to the saturation of the machine responsible to degradation of dsRNA. The immune system cannot recognize these mutations due to preservation of the amino acid sequences. Formation of RNA hairpins or other 2D and 3D DNA structures would lead to the activation of the protein machineries responsible for processing of small RNAs, namely, Drosha (in the nucleus), Dicer and RISC (in the cytoplasm). As a result, in the cytoplasm would have to appear a large number of dsRNA that are not sensitive to conventional RNAases and would have to expel from the cells through exosomes or digested in the autophagosomes. During tumorigenesis cells would extrude short dsRNAs into the extracellular fluid and then into blood. According to the hypothesis, gradual accumulation of synonymous mutations in somatic cells, would lead to the appearance of possibilities for hybridization of RNA. Because the mammalian genome has several degrees of protection from a single mutation, the phenotypic manifestations of mutations in long time would not be no seen. Due to this, a phenotype also will not be changed after most non-synonymous mutations, since the substitution of one amino acid by its homologue in those areas that are not conservative (as most of them), does not change the protein function. Only when the threshold of sensitivity of cells to mutations is crossed, there will be phenotypic manifestations. The hypothesis explains the established phenomenon of constant present a significant number of mutations in cancer cells, rather than a single mutation. It never contains only one mutation. The fidelity of this hypothesis is confirmed by the fact that the deletion of the machines responsible for processing of dsRNA (Drosha, Dicer or RISC) enhanced the tumorigenicity of cancer cell lines.

The first task of this study is to assess in the human genome possible intra-molecular and inter-molecular RNA hybridization. The second task is the search of the molecular mechanisms responsible for the delivery and concentration of RNA in exosomes and their secretion, as well as mechanisms responsible for exosome fusion with target normal cells and the appearance of double chains of RNA in the blood. The final result of the project could be the development of the early test of cancer, namely, the elevation of double stranded RNA concentration in blood. In the case of success, a completely new era of early tumours diagnosis will arise – it will be possible to identify double-stranded RNA helices in the blood at the very early stages of the tumour formation.

Other current projects

  1. Role of Golgi glycosylation enzymes in tumorigenesis. The group is also interested in deciphering of the mechanisms responsible for the concentration, transport of and glycosylation within the GA and to identify among them those related to tumorigenesis.
  2. Development of the models of intracellular transport and in particular the model of intra/Golgi transport able to explain all literature experimental data together with the correct mapping of functions of proteins involved into intracellular transport.
  3. Examination of morphological alterations induced by deletion or over expression of proteins involved in intra-Golgi transport.
  4. Cystic fibrosis as a model of transport of unconventional cargoes along the secretory pathway and in particular intra–Golgi transport.

 

update: February 2011
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