KRAS and BRAF mutations appear of relevance in the genesis and progression of several solid tumor types but the co-occurrence and interaction of these mutations have not yet been fully elucidated. ...Using a microsatellite stable (MSS) colorectal cancer (CRC) cell line (Colo741) having mutated BRAF and KRASWT, we also aimed to investigate the KRAS-BRAF interaction. Gene expression profiles for control KRASWT, KRAS G12V and KRAS G12D transfected cells were obtained after cell clone selection and RT-PCR screening. Extensive qPCR was performed to confirm microarray data.
We found that the KRAS G12V state deregulated several genes associated to cell cycle, apoptosis and nitrogen metabolism. These findings indicated a reduced survival and proliferation with respect to the KRASWT state. The KRAS G12D state was, instead, characterized by several other distinct functional changes as for example those related to chromatin organization and cell-cell adhesion without affecting apoptosis related genes.
These data predict that the G12D mutation may be more likely selected in a BRAF mutated context. At the same time, the presence of the KRAS G12V mutation in the cells escaping apoptosis and inducing angiogenesis via IL8 may confer a more aggressive phenotype. The present results get along with the observations that CRCs with G12V are associated with a worse prognosis with respect to the WT and G12D states and may help identifying novel CRC pathways and biomarkers of clinical relevance.
The mucosae of the oral cavity are different at the histological level but appear all equally exposed to common genotoxic agents. As a result of this exposure, changes in the mucosal epithelia may ...develop giving rise to Oral Potentially Malignant Lesions (OPMLs), which with time may in turn progress to Oral Squamous Cell Carcinomas (OSCCs). Therefore, much effort should be devoted to identify features able to predict the likeliness of progression associated with an OPML. Such features may be helpful in assisting the clinician to establish both appropriate therapies and follow-up schedules. Here, we report a pilot study that compared the occurrence of DNA aneuploidy and chromosomal copy number aberrations (CNAs) in the OPMLs from different oral anatomical subsites.
Samples from histologically diagnosed OPMLs were processed for high resolution DNA flow cytometry (hr DNA-FCM) in order to determine the relative DNA content expressed by the DNA index (DI). Additionally, array-Comparative Genomic Hybridization (a-CGH) analysis was performed on DNA obtained from diploid nuclei suspensions directly. When aneuploid nuclei were detected, these were physically separated from diploid nuclei on the base of their DI values by means of a DNA-FCM-Sorter in order to improve the a-CGH analysis.
Tongue OPMLs were more frequently associated with DNA aneuploidy and CNAs than OPMLs arising from all the other mucosal subsites.
We suggest that the follow-up and the management of the patients with tongue OPMLs should receive a distinctive special attention. Clearly, this hypothesis should be validated in a prospective clinical study.
γ-secretase inhibitors have been proposed as drugs able to kill cancer cells by targeting the NOTCH pathway. Here, we investigated two of such inhibitors, the Benzyloxicarbonyl-Leu-Leu-Nle-CHO ...(LLNle) and the N - N -(3,5-difluorophenacetyl)- l -alanyl- S -phenylglycine t -butyl ester (DAPT), to assess whether they were effective in killing human glioblastoma tumor–initiating cells (GBM TIC)
in vitro . We found that only LLNle was able at the micromolar range to induce the death of GBM TICs by apoptosis. To determine the
cellular processes that were activated in GBM TICs by treatment with LLNle, we analyzed the amount of the NOTCH intracellular
domain and the gene expression profiles following treatment with LLNle, DAPT, and DMSO (vehicle). We found that LLNIe, beside
inhibiting the generation of the NOTCH intracellular domain, also induces proteasome inhibition, proteolytic stress, and mitotic
arrest in these cells by repressing genes required for DNA synthesis and mitotic progression and by activating genes acting
as mitotic inhibitors. DNA content flow cytometry clearly showed that cells treated with LLNle undergo arrest in the G 2 -M phases of the cell cycle. We also found that DAPT and L-685,458, another selective Notch inhibitor, were unable to kill
GBM TICs, whereas lactacystin, a pure proteasome inhibitor, was effective although at a much less extent than LLNle. These
data show that LLNle kills GBM TIC cells by inhibiting the proteasome activity. We suggest that LLNle, being able to target
two relevant pathways for GBM TIC survival, may have a potential therapeutic value that deserves further investigation in
animal models. (Mol Cancer Res 2009;7(11):1822–34)
J Oral Pathol Med (2012) 41: 119–123
Objective: ‘Field cancerization’ is an accepted model for oral carcinogenesis. So far, genetically altered fields have been just reported in the presence of ...carcinomas. This study assessed the distant mirror fields (MFs) of oral precancer by DNA high‐resolution flow cytometry (hr DNA‐FCM) and array‐Comparative Genomic Hybridization (a‐CGH).
Methods: Five leukoplakias without dysplasia (OLs), seven dysplastic leukoplakias (DOLs), and 12 corresponding visually normal and non‐dysplastic MFs were analyzed. DNA aneuploidy (DNA Index, DI ≠ 1) was detected by hr DNA‐FCM on DAPI stained nuclei suspensions. The epithelial DNA aneuploid subclones were FCM‐sorted to obtain genomic DNA for a‐CGH.
Results: Mirror fields, OLs, and DOLs showed increasing prevalence of DNA aneuploidy of, respectively, 8%, 20%, and 57%. The average number of chromosome aberrations (Ch‐Abs) was 2.8 in MFs, 3 in OLs, and 10.6 in DOLs. MFs relative to OLs and DOLs had average numbers of Ch‐Abs, respectively, of 1.8 and 3.6. Ch‐Abs were also observed in DNA diploid sublines, and often the same aberrations were observed in both MFs and corresponding OLs/DOLs.
Conclusion: DNA aneuploidy and Ch‐Abs in MFs, the last ones being mainly gains, indicate an early onset of field effect in oral carcinogenesis.
DNA aneuploid sublines in sporadic colorectal cancers (CRCs) are quite frequent (about 85%) and likely the consequence of chromosomal instability and DNA copy number aberrations (CNAs). In order to ...gain insight into the mechanisms of the diploid-aneuploid transition in CRCs, we compared the CNA status in both diploid and aneuploid sublines. We used fresh/frozen material from 17 aneuploid CRCs, which was separated into 17 DNA diploid and 17 aneuploid sublines using enrichment of the epithelial component by multiparameter flow cytometry and sorting. CNA status of both sublines was obtained by array comparative genomic hybridization. The DNA diploid sublines from the aneuploid CRCs showed already CNAs, in particular, gains at 20 p and 20 q. The same aberrations were detected at increased frequencies in the corresponding DNA aneuploid sublines. Moreover, the very frequent gains/losses of chromosomes 4, 7, 8, 13, 15, and 18 in the DNA aneuploid sublines were absent or rare in the DNA diploid sublines from the same sporadic aneuploid CRCs. The comparison of the DNA diploid and aneuploid sublines from aneuploid CRCs suggests that 20 p and 20 q gains may play a role in the diploid-aneuploid transition. The 20 q chromosomal arm appears of particular interest since it harbors several genes implicated in chromosomal instability.
This prospective observational study investigated the determinants of malignant transformation (MT) in localized oral leukoplakia (OL) and proliferative verrucous leukoplakia (PVL).
Demographic, ...clinical, histological, and DNA ploidy status data were collected at enrolment. Survival analysis was performed (MT being the event of interest).
One-hundred and thirty-three patients with OL and 20 patients with PVL entered the study over 6 years (mean follow-up 7.8 years). The presence of OED, DNA ploidy, clinical presentation, and lesion site were associated with MT in patients with OL in a univariate analysis. In a multivariate model, OED was the strongest predictor of MT in patients with OL. Adding DNA ploidy increased the model's predictive power. None of the assessed predictors was associated with MT in patients with PVL.
DNA ploidy might identify a subset OL with low risk or minimal risk of MT, but it does not seem to be a reliable predictor in patients with PVL.
Most patients affected by Glioblastoma multiforme (GBM, grade IV glioma) experience a recurrence of the disease because of the spreading of tumor cells beyond surgical boundaries. Unveiling ...mechanisms causing this process is a logic goal to impair the killing capacity of GBM cells by molecular targeting.We noticed that our long-term GBM cultures, established from different patients, may display two categories/types of growth behavior in an orthotopic xenograft model: expansion of the tumor mass and formation of tumor branches/nodules (nodular like, NL-type) or highly diffuse single tumor cell infiltration (HD-type).
We determined by DNA microarrays the gene expression profiles of three NL-type and three HD-type long-term GBM cultures. Subsequently, individual genes with different expression levels between the two groups were identified using Significance Analysis of Microarrays (SAM). Real time RT-PCR, immunofluorescence and immunoblot analyses, were performed for a selected subgroup of regulated gene products to confirm the results obtained by the expression analysis.
Here, we report the identification of a set of 34 differentially expressed genes in the two types of GBM cultures. Twenty-three of these genes encode for proteins localized to the plasma membrane and 9 of these for proteins are involved in the process of cell adhesion.
This study suggests the participation in the diffuse infiltrative/invasive process of GBM cells within the CNS of a novel set of genes coding for membrane-associated proteins, which should be thus susceptible to an inhibition strategy by specific targeting.Massimiliano Monticone and Antonio Daga contributed equally to this work.
Mtfr1/Chppr is a nuclear gene coding for a mitochondrial protein capable of inducing fission of this organelle in a sequence-specific manner. Here we show that in mice, Mtfr1/Chppr is ubiquitously ...expressed and displays the highest level of expression in pubertal and adult testes and in particular in spermatids and Leydig cells. To investigate Mtfr1 function in vivo, we analyzed homozygous mice null for this gene obtained through a gene trap approach. We show that these mice fail to express Mtfr1 and that in their testes several genes coding for enzymes involved in the defense against oxidative stress are downregulated. Among these, we studied in particular glutathione peroxidase 3 and show its expression in selected testis cell types. Furthermore, we demonstrate oxidative DNA damage specifically in testes of Mtfr1-deficient mice likely resulting from a reduced antioxidant activity. As a whole, these data suggest that Mtfr1 protects the male gonads against oxidative stress.
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Neratinib (NE) is an irreversible pan-ERBB tyrosine kinase inhibitor used to treat breast cancers (BCa) with amplification of the ERBB2/HER2/Neu gene or overexpression of the ERBB2 ...receptor. However, the mechanisms behind this process are not fully understood. Here we investigated the effects of NE on critical cell survival processes in ERBB2+ cancer cells. By kinome array analysis, we showed that NE time-dependently inhibited the phosphorylation of two distinct sets of kinases. The first set, including ERBB2 downstream signaling kinases such as ERK1/2, ATK, and AKT substrates, showed inhibition after 2 h of NE treatment. The second set, which comprised kinases involved in DNA damage response, displayed inhibition after 72 h. Flow cytometry analyses showed that NE induced G0/G1 cell cycle arrest and early apoptosis. By immunoblot, light and electron microscopy, we revealed that NE also transiently induced autophagy, mediated by increased expression levels and nuclear localization of TFEB and TFE3. Altered TFEB/TFE3 expression was accompanied by dysregulation of mitochondrial energy metabolism and dynamics, leading to a decrease in ATP production, glycolytic activity, and a transient downregulation of fission proteins. Increased TFEB and TFE3 expression was also observed in ERBB2-/ERBB1 + BCa cells, supporting that NE may act through other ERBB family members and/or other kinases. Overall, this study highlights NE as a potent activator of TFEB and TFE3, leading to the suppression of cancer cell survival through autophagy induction, cell cycle arrest, apoptosis, mitochondrial dysfunction and inhibition of DNA damage response.
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