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WO2008138868A2 - Diagnostic et/ou pronostic de cancer in vitro par analyse de l'expression de la protéine gtpase - Google Patents

Diagnostic et/ou pronostic de cancer in vitro par analyse de l'expression de la protéine gtpase Download PDF

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WO2008138868A2
WO2008138868A2 PCT/EP2008/055714 EP2008055714W WO2008138868A2 WO 2008138868 A2 WO2008138868 A2 WO 2008138868A2 EP 2008055714 W EP2008055714 W EP 2008055714W WO 2008138868 A2 WO2008138868 A2 WO 2008138868A2
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rho
expression
cancer
prognosis
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WO2008138868A3 (fr
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Mª Teresa GÓMEZ DEL PULGAR
Ana RAMÍREZ DE MOLINA
Juan Carlos LACAL SANJUÁN
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Consejo Superior de Investigaciones Cientificas CSIC
Traslational Cancer Drugs Pharma SL
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Traslational Cancer Drugs Pharma SL
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the present invention can be included within several fields such as Genetic Engineering, Biotechnology and Pathology. Specifically, the present invention relates to a method of in vitro diagnosis and/or prognosis of breast cancer and/or lung cancer by the analysis of Rho-family GTPase protein expression.
  • Rho-family GTPases belong to the large superfamily of Ras proteins, with which they share a high homology of sequence and structure. There are a total of 22 members which can be divided in turn into six groups in accordance with the sequence homology, the structural motifs and the biological function. These subfamilies are Rho (RhoA, RhoB, RhoC), Rac (Racl, Rac2, Rac3, RhoG), Cdc42 (Cdc42, TClO, TCL, Chp, Wrch-1), Rnd (Rndl, Rnd2, Rnd3/RhoE), RhoBTB (RhoBTBl and RhoBTB2) and Miro (Miro-1 and Miro-2).
  • RhoD Three of the members of the family, RhoD, Rif and RhoH/TTF are not grouped in any of the following subgroups (Wennerberg and Der, 2004).
  • the most atypical members are the RhoBTB and Miro subfamilies, since these proteins are considerably larger than the classic GTPases and contain structural domains not present in other Rho GTPases.
  • Rho proteins mediates a wide spectrum of cell activities among which we can highlight the regulation of proliferation and apoptosis processes, organization of the actin cytoskeleton, control of gene expression and membrane traffic. It is therefore not surprising that the Rho proteins are involved in the tumourigenesis process at several levels (reviewed in (Gomez del Pulgar et al, 2005). Their activity is directly related to cell proliferation, the regulation of the cell cycle, cell adhesion and motility, and the degradation of the basal membrane of the original tissue for the extravasation to the blood stream and its later intravasation in a distant tissue.
  • Rho GTPases are guanine nucleotide -binding proteins which cycle between an active state, bound to GTP, and an inactive state, bound to GDP. This cycle is regulated by three distant families of proteins, the guanine exchange factors (GEFs), the activating proteins of the intrinsic activity of hydrolysis of GTP to GDP (GTPase activating proteins, GAPs), and the guanine dissociation inhibitors (GDIs). After the loading of GTP, the Rho proteins suffer a formational change which allows them to interact with several effectors which are in charge of propagating the signal to inside the cell.
  • GEFs guanine exchange factors
  • GAPs GTPase activating proteins
  • GDIs guanine dissociation inhibitors
  • Rho GTPases The deregulation of the expression and the activity of some of the best known Rho GTPases, and of some of their most studied effects, has a very important role in human carcinogenesis (Aronheim et al, 1998; Gomez del Pulgar et al, 2005).
  • Rho proteins The relevance of the rest of the family of Rho proteins, as well as of other effectors, in tumourigenesis has not yet been defined.
  • the present invention centred on the analysis of the expression of the three genes Rnd, Rndl, Rnd2 and RhoE/Rnd3 in tumour tissues. These proteins constitute a subgroup of the Rho family with unusual biochemical properties compared with those of other family members.
  • the Rnd subfamily is farnesylated and suggests that the interaction of the Rnd proteins with membranes is probably different from other members of the family.
  • the members of the Rnd subfamily have a specific tissue expression pattern.
  • the results of a Northern blot in human tissues indicated that the mRNA expression of these three genes is different according to the tissue analysed.
  • Rndl is largely expressed in the brain, liver and in smaller quantities in other tissues;
  • Rnd2 is largely found in the testicles and
  • Rnd3/RhoE is expressed in practically all the tissues at low levels, although their expression levels significantly vary among different cell types (Chardin, 2006).
  • the present invention relates to an in vitro method of diagnosis / prognosis of breast cancer and/or lung cancer by the analysis in a single sample of the expression of the Rnd2/Rho 7 protein.
  • a 2nd preferred embodiment of the present invention relates to an in vitro method of diagnosis / prognosis of breast cancer and/or lung cancer by the analysis in a single sample and simultaneously, of the expression profile of the subfamily of Rnd proteins, comprised of the following proteins: Rndl / Rho 6, Rnd2 / Rho 7 and Rnd3 / RhoE / Rho8.
  • document Dl is the closest to the object of the invention as it analyses the expression levels and distribution of Rho family GTPases (Rho-A, B, C and G, Rho-6, 7 and 8) and of guanine dissociation inhibitors (GDIs), (Rho-GDI-beta and Rho- GDI-gamma), in breast cancer and their evaluation with regard to the prognostic value.
  • Rho family GTPases Rho-A, B, C and G, Rho-6, 7 and 8
  • GDIs guanine dissociation inhibitors
  • Rho-GDI-beta and Rho- GDI-gamma guanine dissociation inhibitors
  • the present invention reflects a change in the expression level of said Rho-7 and Rho-8, proteins in the tumour tissues, with respect to the normal tissues, the degree of expression of these two markers being what determines the cancer diagnosis or prognosis.
  • Document D2 does not relate to GTPases of the invention object of the present invention but describes in it the identification of Rho C as breast cancer marker. It demonstrates that the overexpression of Rho C induces the malign transformation of immortalized human breast epithelial cells, producing an aggressive phenotype, which is very mobile and invasive. Rho C expression in different IBC breast cancers is produced at late stages, whilst the cancerous cells acquire invasive capacity. In IBC, the most lethal type, Rho C appears at early stages.
  • Rho C seems to be a marker of the metastastic potential in breast cancer.
  • the detection of IBC by the Rho C protein may be a useful tool for identifying small invasive ductal carcinomas, with a great probability for producing metastasis.
  • GTPases analysed in the present invention with the prognosis / diagnosis of lung cancer.
  • the objective technical problem resolved by the present invention constitutes the diagnosis of breast cancer and / or lung cancer in a greater percentage of patients than the existing methods, or alternatively to them, as well as a better prognosis of the evolution of said disease, on having new markers of the disease such as the Rnd2/Rho 7 protein, alone or in a combined analysis of the expression profile of the whole subfamily of Rnd GTPase proteins, constituted by Rndl/Rho 6, Rnd2/Rho 7 and Rnd3/RhoE/Rho8.
  • the document which could possibly be considered as closest to the invention is Dl because it analyses the expression and distribution levels of Rho-family GTPases (Rho-A, B, C and G, Rho-6, 7 and 8) and of guanine dissociation inhibitors (GDIs), (Rho-GDI-beta and Rho- GDI-gamma), in breast cancer and their evaluation in terms of prognostic value. Therefore, said document Dl discloses the fact that Rho 6 is overexpressed in breast cancer. But, with regard to Rho-7 and Rho-8, document Dl says that the level of said proteins is similar in the tumour and normal tissues.
  • the present invention reflects a change in the expression level of said Rho-7 and Rho-8 proteins in tumour tissues, with respect to normal tissues, the degree of expression of these two markers being what determines the prognosis.
  • the common part between the invention and Dl is that both diagnose breast cancer as they detect Rndl overexpression.
  • Dl does not go beyond this, it being the work of the present invention to make it possible to discover the prognosis or evolution of the disease in accordance with the degree of Rnd2 and Rnd3 expression, which makes it possible to diagnose or make a prognosis of the development of the cancer in a greater number of patients that with the marker described in Dl would not be detected.
  • the present invention has the main objective of the analysis of the gene expression levels of Rho-family GTPases (Table 6), specifically of the members of the Rnd subfamily: Rndl, Rnd2 and Rnd3, either specifically for one of its members, the Rnd2 protein, or simultaneously in a same sample of tumour tissue, of all them and its use for the diagnosis and / or prognosis of cancer.
  • Said analysis was particularly developed in two of the currently most relevant human tumours: breast cancer and lung cancer, in order to identify new effective markers in the early diagnosis of cancer, the staging and the prognosis.
  • the present invention relates to an in vitro method of diagnosis / prognosis of breast cancer and/or lung cancer by the analysis in a single sample of the expression of the Rnd2/Rho 7 protein.
  • a 2nd preferred embodiment of the present invention relates to an in vitro method of diagnosis / prognosis of breast cancer and/or lung cancer by the analysis in a single sample and simultaneously, of the expression profile of the subfamily of Rnd proteins, comprised of the following proteins: Rndl / Rho 6, Rnd2 / Rho 7 and Rnd3 / RhoE / Rho8.
  • the present invention also relates to a kit or test device which comprises at least sequences of nucleic acids capable of hybridizing with sequences belonging to the Rnd 2/ Rho 7 gene, specifically, combined with sequences of nucleic acids capable of hybridizing also simultaneously in a single sample with gene sequences of the genes Rndl/Rho6 and/or Rnd 3/Rho 8, or combinations thereof, for the in vitro diagnosis / prognosis of cancer which comprises the analysis in a single sample either of the expression level of the gene Rnd2/Rho 7, or, simultaneously, of the expression profile of said genes or of combinations thereof.
  • an embodiment of the present invention relates to a method of in vitro diagnosis / prognosis of cancer, principally of breast cancer and/or lung cancer by the analysis in a single sample of either the expression profile of the Rnd2/Rho 7 protein, or, simultaneously, of the expression profile of the subfamily of Rnd proteins, comprised of the following proteins: Rndl / Rho 6, Rnd2 / Rho 7 and Rnd3 / RhoE / Rho8, to the expression profile of these proteins it is possible to add the measurement of the expression profile corresponding to other GTPases of other subfamilies, such as: Rho, Rac, Cdc42, RhoBTB and Miro.
  • the invention further comprises a test device which comprises sequences of nucleic acids capable of hybridizing with sequences belonging to the genes of the different families of GTPases whose expression profile one wants to measure.
  • the use of these test devices further permits diagnosing cancer, making a prognosis of the development of the disease, as well as a monitoring of the treatments for its cure, or of the phases of development wherein the disease is found at any time.
  • FIG. 1 Analysis of Rnd2 expression in breast cancer samples.
  • the y-axis (Y) represents the expression level of Rnd2 and the x-axis (X) represents the different tumour samples with N being the normal samples M each one of the tumour samples (A), tumour size (B), the level of lymph gland invasion (C), the histological grade (D) and the relapse
  • Figure 2 A.
  • the y-axis represents cumulative survival and the x-axis represents overall survival in months, in patients with breast cancer in accordance with Rnd2 expression.
  • the y-axis represents cumulative survival and the x-axis represents disease-free survival in months, in patients with breast cancer in accordance with Rnd2 expression. (+) cases wherein Rnd2 expression is equal to or greater than the expression in normal tissue (upper curve).
  • the y-axis represents the expression level of RhoE/Rnd3 and the x-axis represents the different tumour samples with N being the normal samples and M each one of the tumour samples (A), the age of the patient (B), tumour size (C), the level of lymph gland invasion
  • the y-axis represents cumulative survival and the x-axis represents overall survival in months, in patients with breast cancer in accordance with RhoE/Rnd3 expression.
  • the y-axis represents cumulative survival and the x-axis represents disease-free survival in months, in patients with breast cancer in accordance with RhoE/Rnd3 expression.
  • FIG. B Kaplan-Meier curve
  • Figure 5 Analysis of the Rndl expression levels in lung cancer samples.
  • the y-axis represents the expression level of Rndl in absolute quantity (AQ) and the x-axis represents the different tumour samples with N being the normal samples L each one of the tumour samples (A), tumour size (B), level of lymph gland invasion (C) and the relapse (D).
  • C Average value of Rndl expression in accordance with the level of lymph gland invasion.
  • D Average value of Rndl expression in accordance with the relapse due to appearance ofmetastasis.
  • Rndl expression is analysed comparing the values of the averages in accordance with the clinical-pathological parameter.
  • the relation between the mRNA levels of the Rndl gene and the clinical-pathological parameters were based on the non- parametric Mann Whitney U test. It is observed that Rndl expression is not significantly associated to any of the parameters under study.
  • the y-axis represents cumulative survival and the x-axis represents overall survival in months, in patients with lung cancer in accordance with Rndl expression.
  • the y-axis represents cumulative survival and the x-axis represents disease-free survival in months, in patients with lung cancer in accordance with Rndl expression.
  • FIG. 7 Analysis of Rnd2 expression in lung cancer samples.
  • the y-axis (y) represents the expression level of Rnd2 in absolute quantity (AQ) and the x-axis (X) represents the different tumour samples with N being the normal samples and L each one of the tumour samples (A), tumour size (B), the level of lymph gland invasion (C) and the relapse (D).
  • the y-axis represents cumulative survival and the x-axis represents overall survival in months, in patients with lung cancer in accordance with Rnd2 expression.
  • the y-axis represents cumulative survival and the x-axis represents disease-free survival in months, in patients with lung cancer in accordance with Rnd2 expression.
  • cut-off point the value 10.5 (which is a point with the same sensitivity as the value of the median, of 73%, but with greater specificity, 66%, according to analysis of the ROC curve). (+) cases wherein Rnd2 expression is greater than the cut-off point (lower curve).
  • FIG. 9 Analysis of the expression levels of RhoE/Rnd3 in lung cancer samples.
  • the y-axis represents the expression level of RhoE/Rnd3 and the x-axis represents the different tumour samples with N being the normal samples and L each one of the tumour samples (A), tumour size (B), the level of lymph gland invasion (C) and the relapse (D).
  • tumour samples have an expression level greater than the expression in normal tissue (the expression average is 7.48, median 5.43, the normal tissue expresses 2.12, the pool of normal tissues 1.67).
  • the averages are compared in accordance with the clinical-pathological parameters, no significant differences are found.
  • the present invention relates to an in vitro method of diagnosis / prognosis of breast cancer and/or lung cancer by the analysis in a single sample of the expression of the Rnd2/Rho 7 protein.
  • a 2nd preferred embodiment of the present invention relates to an in vitro method of diagnosis / prognosis of breast cancer and/or lung cancer by the analysis in a single sample and, simultaneously, of the expression profile of the subfamily of Rnd proteins, comprised of the following proteins: Rndl / Rho 6, Rnd2 / Rho 7 and Rnd3 / RhoE / Rho8.
  • the study was developed in two of the currently most relevant types of cancer: breast cancer and lung cancer, with the aim of identifying new effective markers in the early diagnosis of these tumours, the staging and the prognosis.
  • Rho family genes as well as of its main effectors, is carried out by a quantitative PCR study in real time.
  • the extraction of total RNA samples from the tissue samples is necessary, as well as their processing for the PCR quantification in real time of the messenger RNA levels (mRNA) of each gene.
  • mRNA messenger RNA levels
  • the breast and lung tissue samples belong to patients who have undergone surgery in the Hospital de La Paz, Madrid. None of the patients received adjuvant therapy prior to surgery. All the samples were collected at the time of resection and were immediately frozen in liquid nitrogen. The ethical committee of the Hospital La Paz approved this study.
  • the breast cancer samples are of infiltrative ductal histology type and correspond to patients with ages between 27 and 80 years of age (average 57 years of age). The median of the monitoring time was 70 months (range: 25-126). At the time of analysis, 12 of the patients had suffered relapse due to the disease, whilst the rest had not relapsed.
  • the pathological parameters of the piece such as the degree of differentiation, the size (pT) and the data on regional lymph gland invasion (pN) are set down in Table 1, as well as the clinical data regarding relapse of the disease.
  • RNA of the tissue samples was extracted using Trizol Reagent (Invitrogen) and then passing the samples through the RNeasy Mini kit (Qiagen), according to the manufacturer's instructions.
  • the RNeasy Mini kit from Qiagen enables the extraction from small quantities of material. It is based on the selective bonding properties of the RNA to a silica-gel membrane and the contaminants can be eliminated by washing.
  • the integrity of each one of the RNA samples was evaluated by electrophoresis in agarose gel and the samples were quantified with the Nanodrop spectrophotometer for the later retrotranscription reaction.
  • RNA sample was carried out from 1 g of total RNA with the High-Capacity cDNA Archive Kit (Applied Biosystems). This kit contains all the necessary components, including a buffer optimized for the reaction, dNTPs, random primers and the reverse transcriptase MultiScribeTM MuLV.
  • the amplification products are observed as the PCR cycles elapse. It is based on the detection and quantification of a fluorescent Reporter, whose signal increases in direct proportion to the quantity of PCR product in the reaction.
  • the data are acquired when the amplification is still in exponential phase.
  • the data collected corresponds to the cycle number wherein the fluorescence exceeds the established threshold, on increasing the emission intensity of the fiuorochrome with respect to the Background noise.
  • This cycle number is called Ct: Threshold Cycle.
  • the Ct is determined in the exponential phase of the reaction and is more reliable than the conventional end point measurements.
  • the Ct is inversely proportionate to the number of copies of the initial target: the greater the concentration the less Ct measured.
  • the threshold set must be the same for all measurements.
  • Density Arrays have been used for the amplification of the cDNA. It is a microfluidic card which contains the specific probes of each gene deposited in each one of the 384 wells, with a configuration selected according to the number of replicas desired of each gene. In the present study, the configuration chosen contained the genes selected in duplicate.
  • the amplification of an endogenous control is used to standardize the quantity of RNA.
  • the gene of glyceraldyde-3 -phosphate dehydrogenase (GAPDH), or 18S ribosomal RNA (rRNA) were used as endogenous controls.
  • the probes deposited in each well of the card are chosen from those available in Applied Biosystems for our genes of interest.
  • the probes are called TaqMan® Gene Expression Assays (Applied Biosystems) and are designed to recognize a region without ambiguities in its sequence, without polymorphisms or repeated sequences. Whenever possible, the tests are designed in the exon-exon connections, which avoid the co- amplification of contaminant genomic DNA.
  • the probes chosen in the present study are specified in Tables 3, 4.
  • the microfluidic technology uses 8 loading doors, each connected to 48 reaction chambers. This characteristic reduces the number of pipetting steps.
  • 50 nanograms of cDNA are combined with the Taqman Universal Master Mix (Applied Biosystems) per loading door, the card is briefly centrifuged and it is sealed.
  • the Array is taken to 50 0 C for 2 min and 95 0 C 10 min, followed by 40 cycles at 95 0 C for 15 s, and 60 0 C 1 min.
  • the amplification data are collected using the SDS 2.1 software from Applied Biosystems, and analysed with the comparative method of relative quantification.
  • the reference sample is a commercial RNA of normal breast tissue for the breast tissue samples. In the lung study, a commercial RNA from normal lung tissue and a pool of normal tissues adjacent to some tumours under study have been used. The commercial RNAs are from Stratagene. To calculate the relative expression of each gene, the 2 " Ct method was used
  • RQ Relative Quantity
  • Rho-family GTPase genes The relation between the mRNA expression levels of Rho-family GTPase genes and the clinical-pathological factors was analysed statistically.
  • the continuous variables with abnormal distribution were compared using the Mann Whitney and Kruskal-Wallis U-test.
  • To analyse the continuous variables with normal distribution, the t-test was used.
  • the survival curves were made according to the Kaplan-Meier method and the significance was evaluated with the log rank test.
  • the quantification of the linear relation between the two variables was studied by calculating Spearman's non-parametric coefficient of correlation. All calculations were made with the SPSS software, version 13. The differences were considered statistically significant when the value o ⁇ p was lower than 0.05.
  • the expression profile of Rnd2 of these tumour samples is very variable compared with normal tissue (Fig. IA), which led to trying to group the samples in accordance with the clinical-pathological parameters and analysing the average expression value in accordance with tumour size (Fig. IB), the capacity of invading lymph glands (Fig. 1C), the histological grade (Fig. ID) and the relapse of the patient due to the disease (Fig. ID). For any of these parameters, a tendency is observed for a reduction in Rnd2 expression, the loss of expression with a greater histological grade being statistically significant (p ⁇ 0.05).
  • the Rnd2 gene is the closest to BRCAl (Smith et al, 1996), a gene whose mutations increase susceptibility to breast and ovarian cancer. Rnd2 is located in opposite orientation to BRCAl. However, there are no data on the effects of the deletion of the gene or translocations in breast cancer (Smith et al., 1996).
  • RhoE expression is a factor of bad prognosis in breast cancer, since the reduced levels of this gene in comparison with normal tissue is associated to a greater tumour size and to the relapse of the patient.
  • RhoE This reduction or lack of RhoE expression indicates that it behaves as a tumour suppressor gene in breast cancer since RhoE expression may be critical in preventing the transformation of epithelial breast cells.
  • RhoE antagonizes the RhoA GTPase, which is overexpressed and activated in different human tumours, among those of the breast (reviewed in Gomez del Pulgar et al, 2005), RhoE could counteract the action of RhoA to maintain the cell growth processes and normal differentiation in breast tissue.
  • RhoE/Rnd3 is a tumour suppressor gene and that its deregulation may be a marker of tumour progression, in accordance with its antagonist role of RhoA and as cell cycle inhibitor.
  • Rndl mRNA expression levels in lung cancer mRNA expression levels of Rnd genes in lung cancer are expressed as the absolute quantity (AQ) of the Rnd gene in a tumour sample once normalized against the 18S ribosomal RNA endogenous control gene. As a consequence, it indicates the expression of mRNA of the gene in a sample of normal commercial tissue (Stratagene, catalogue no. 540019). It also represents mRNA expression in the pool of normal tissues included in the study.
  • Fig. 5 shows the results of the Rndl mRNA levels in lung cancer samples.
  • the average of Rndl expression is 1.57, and the median 0.99, the quantity of the gene in the normal commercial tissue (Stratagene) being 1.63.
  • the normal pool of tissues has a quantity of 1.87).
  • the disease-free survival was determined as the interval between the time of diagnosis and the detection of the first relapse.
  • the value of the median 0.99, (with a sensitivity of 82% and specificity of 58% according to analysis of the ROC curve). This value represents a reduction in expression of 1.6 times below the expression in normal tissue.
  • FIG. 7A shows mRNA expression levels of the Rnd2 gene in lung cancer samples.
  • the expression average is 20.9 and the median 9.06, the quantity of mRNA in the normal tissue being 5.62. If we compare the expression averages in accordance with the clinical- pathological parameters, there are no significant differences (Fig. 7B, 7C and 7D).
  • RhoE/Rnd3 expression in each sample is indicated in Fig. 9A, observing that most of the tumour samples have an expression level higher than expression in normal tissue (the average expression is 7.48, median 5.43, the normal tissue expresses 2.12, the pool of normal tissues 1.67).
  • Fig. 9B, 9C and 9D When we compare the averages in accordance with the clinical- pathological parameters (Fig. 9B, 9C and 9D), no significant differences are found.
  • Rndl / Rho 6 is an oncogen which is overexpressed 100% of the times in the malignant cells of breast tissue and therefore can be used to diagnose breast cancer.
  • Rnd2 / Rho 7 behaves as a tumour suppressor since the results of the research suggest that the loss of Rnd2 expression is associated to the worst prognosis of the disease. Particularly well-known is the observation made with respect to the histological grade and to the disease-free survival on finding a statistically significant relation between the decrease in Rnd2 expression, the increase in histological grade (the lower levels of Rnd2 are located in histological grade III) and the decrease in disease-free survival. On analysing the overall survival practically, the association between the loss of Rnd2 expression and a less overall survival is significant. Therefore, Rnd2 can be considered as prognosis marker, their low levels being symptoms of bad prognosis.
  • Rndl expression is homogeneous, it therefore being possible to behave as a marker useful for the diagnosis of breast cancer, the degree of expression of the other two markers (Rnd2 and Rnd3) being those which determine the prognosis or evolution of the disease.
  • the present invention relates in a first aspect to the method of in vitro diagnosis and/or prognosis of cancer which comprises the analysis of the expression profile of the Rnd 2/ Rho 7 or Rnd 3 /RhoE/ Rho 8 proteins, where the cancer analysed is breast cancer or lung cancer.
  • the present invention relates in a second aspect to a method of in vitro diagnosis and/or prognosis of cancer which comprises the analysis of the expression profile of the
  • Rnd 2/ Rho 7 or Rnd 3 /RhoE/ Rho 8 proteins where the cancer analysed is breast cancer or lung cancer, which further comprises the analysis of the expression level of one of the
  • Rho subfamily GTPases belonging to one of the following subfamilies of GTPases: Rho subfamily
  • Rho A, Rho B and Rho C (comprised of Rho A, Rho B and Rho C), Rac subfamily (comprised of Racl, Rac2, Rac3 and Rho G), Cdc42 subfamily (comprised of Cdc42, TClO, TCL, Chp and Wrch-1), Rho BTB subfamily (comprised of RhoBTB 1 and RhoBTB2), Miro subfamily (comprised of: Miro-1 and Miro-2) or the analysis of the expression level of one of the GTPases belonging to the group comprised of: Rho D, Rif and RhoH/TTF.
  • the present invention relates in a third aspect to a kit which comprises at least gene sequences capable of hybridizing with sequences belonging to the genes Rnd 2/ Rho 7 or Rnd 3 / Rho 8, for the in vitro diagnosis / prognosis of cancer where the cancer analysed is breast cancer or lung cancer.
  • the present invention relates in a fourth aspect to a kit which comprises at least gene sequences capable of hybridizing with sequences belonging to the genes Rnd 2/ Rho 7 or Rnd 3 / Rho 8, for the in vitro diagnosis / prognosis of cancer, where the cancer analysed is breast cancer or lung cancer, which further comprises at least gene sequences capable of hybridizing with one of the sequences belonging to the genes of the GTPases of one of the following subfamilies: Rho subfamily (comprised of Rho A, Rho B and Rho C), Rac subfamily (comprised of Racl, Rac2, Rac3 and Rho G), Cdc42 subfamily (comprised of Cdc42, TClO, TCL, Chp and Wrch-1), RhoBTB subfamily (comprised of RhoBTB 1 and RhoBTB2), Miro subfamily (comprised of: Miro-1 and Miro-2) or gene sequences capable of hybridizing with one of the sequences belonging to the genes of the GTPases comprised in
  • RhoBTB2 Hs00248529 ml NM 015178 RhoBTB2 Related to Rho containing domain BTB, 2
  • PKN HsOOI 77028_m1 NM 002741 PRKCL1 protein kinase similar to C, 1 mDiai HsOOI 93268_m1 NM 005219 DIAPH 1 homologue 1 of the "diaphanous" gene (Drosophila) mDia2 Hs00246501_m1 NM 006729 DIAPH2 homologue 2 of the "diaphanous" gene (Drosophila)
  • Citron Hs00392339_m1 NM 007174 CIT serine/threonine kinase 21 which interacts with rho
  • N-WASP Hs00187614 ml NM 003941 WASL gene similar to Wiskott-Aldrich's syndrome
  • Table 5 Relation between the expression level of mRNA of Rnd2 and each clinical- pathological factor.
  • Rho-family GTPase proteins studied in the present invention are listed in Table 6.
  • Chp a homologue of the GTPase Cdc42Hs, activates the JNK pathway and is implicated in reorganizing the actin cytoskeleton. Curr Biol 8, 1125-8.
  • RhoE binds to ROCK I and inhibits downstream signaling. MoI Cell Biol 23, 4219-29.
  • RhoE function is regulated by ROCK I-mediated phosphorylation. Embo J 24, 1170-80.
  • RhoE inhibits cell cycle progression and Ras-induced transformation. MoI Cell Biol 24, 7829-40.
  • Rho-family GTPases it's not only Rac and Rho (and I like it). J Cell Sci 117, 1301-12.

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Abstract

L'invention concerne un procédé in vitro de diagnostic/pronostic du cancer, principalement du cancer du sein et/ou du cancer du poumon, qui fait appel à l'analyse dans un seul prélèvement du profil d'expression de la protéine Rnd2/Rho 7 ou, simultanément, du profil d'expression d'une protéine de la sous-famille de protéines Rnd composée des protéines suivantes: Rndl / Rho 6, Rnd2 / Rho 7 et Rnd3 / RhoE / Rho8. On peut ajouter au profil d'expression desdites protéines d'autres GTPases d'autres sous-familles, par exemple: Rho, Rac, Cdc42, RhoBTB et Miro. L'invention se rapporte en outre à un dispositif d'essai qui comprend des séquences d'acides nucléiques capables de s'hybrider avec des séquences appartenant aux gènes des différentes familles de GTPases dont on doit mesurer le profil d'expression. L'utilisation de ce dispositif d'essai permet non seulement de diagnostiquer le cancer, mais également d'établir le pronostic du développement de la maladie et de surveiller les traitements visant à la guérir.
PCT/EP2008/055714 2007-05-09 2008-05-08 Diagnostic et/ou pronostic de cancer in vitro par analyse de l'expression de la protéine gtpase Ceased WO2008138868A2 (fr)

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RU2445627C1 (ru) * 2010-09-02 2012-03-20 Учреждение Российской Академии Наук Институт Биологии Гена Ран Способ диагностики немелкоклеточного рака легких и набор для его осуществления

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SG109604A1 (en) * 2003-09-03 2005-03-30 Singapore General Hospital Pte Method and probes for diagnosing a gynaecological condition
US7767417B2 (en) * 2004-04-20 2010-08-03 New York University Prenyl-electrostatic switch, and methods of use
EP1757692A4 (fr) * 2004-04-21 2007-10-31 Daiichi Seiyaku Co GENE CODANT POUR UN FACTEUR D'ECHANGE DE NUCLOTIDE GUANINE LIANT RhoA
MX2007001640A (es) * 2004-08-10 2007-07-25 Univ Cardiff Metodos y kit para el pronostico de cancer de mama.
US7612080B2 (en) * 2004-11-19 2009-11-03 Cincinnati Children's Hospital Medical Center GTPase inhibitors and use thereof for controlling platelet hyperactivity
US7763418B2 (en) * 2005-07-05 2010-07-27 Cytoskeleton, Inc. Detection of Rho proteins

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RU2445627C1 (ru) * 2010-09-02 2012-03-20 Учреждение Российской Академии Наук Институт Биологии Гена Ран Способ диагностики немелкоклеточного рака легких и набор для его осуществления

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