Targeting the "undruggable": RNA-binding proteins in the spotlight in cancer therapy.

Tumors refractory to conventional therapy belong to specific subpopulations of cancer cells, which have acquired a higher number of mutations/epigenetic changes than the majority of cancer cells. This property provides them the ability to become resistant to therapy. Aberrant expression of certain RNA-binding proteins (RBPs) can regulate the sensitivity of tumor cells to chemotherapeutic drugs by binding to specific regions present in the 3´-UTR of certain mRNAs to promote or repress mRNA translation or by interacting with other proteins (including RBPs) and non-coding RNAs that are part of ribonucleoprotein complexes. In particular, an increasing interest in the RBPs involved in chemoresistance has recently emerged. In this review, we discuss how RBPs are not only affected by chemotherapeutic treatments, but also play an active role in therapeutic responses via the direct modulation of crucial cancer-related proteins. A special focus is being placed on the development of therapeutic strategies targeting these RBPs.

RNA-binding proteins: Underestimated contributors in tumorigenesis.

mRNA export, translation, splicing, cleavage or capping determine mRNA stability, which represents one of the primary aspects regulating gene expression and function. RNA-binding proteins (RBPs) bind to their target mRNAs to regulate multiple cell functions by increasing or reducing their stability. In recent decades, studies of the role of RBPs in tumorigenesis have revealed an increasing number of proteins impacting the prognosis, diagnosis and cancer treatment. Several RBPs have been identified based on their interactions with oncogenes or tumor suppressor genes in human cancers, which are involved in apoptosis, the epithelial-mesenchymal transition (EMT), DNA repair, autophagy, cell proliferation, immune response, metabolism, and the regulation of noncoding RNAs. In this review, we propose a model showing how RBP mutations influence tumorigenesis, and we update the current knowledge regarding the molecular mechanism by which RBPs regulate cancer. Special attention is being devoted to RBPs that represent prognostic and diagnostic factors in cancer patients.

Insights into new mechanisms and models of cancer stem cell multidrug resistance.

The acquisition of genetic alterations, clonal evolution, and the tumor microenvironment promote cancer progression, metastasis and therapy resistance. These events correspond to the establishment of the great phenotypic heterogeneity and plasticity of cancer cells that contribute to tumor progression and resistant disease. Targeting resistant cancers is a major challenge in oncology; however, the underlying processes are not yet fully understood. Even though current treatments can reduce tumor size and increase life expectancy, relapse and multidrug resistance (MDR) ultimately remain the second cause of death in developed countries. Recent evidence points toward stem-like phenotypes in cancer cells, promoted by cancer stem cells (CSCs), as the main culprit of cancer relapse, resistance (radiotherapy, hormone therapy, and/or chemotherapy) and metastasis. Many mechanisms have been proposed for CSC resistance, such as drug efflux through ABC transporters, overactivation of the DNA damage response (DDR), apoptosis evasion, prosurvival pathways activation, cell cycle promotion and/or cell metabolic alterations. Nonetheless, targeted therapy toward these specific CSC mechanisms is only partially effective to prevent or abolish resistance, suggesting underlying additional causes for CSC resilience. This article aims to provide an integrated picture of the MDR mechanisms that operate in CSCs' behavior and to propose a novel model of tumor evolution during chemotherapy. Targeting the pathways mentioned here might hold promise and reveal new strategies for future clinical therapeutic approaches.

Absence of Relationship between Mitochondrial DNA Haplogroups and Cisplatin-Induced Hearing Loss.

BACKGROUND: Many drugs used for cancer chemotherapy produce reactive oxygen species, thus leading to various complications including nephrotoxicity, cardiotoxicity, and ototoxicity. OBJECTIVE: We have provided a haplogroup analysis of a cohort of cancer patients treated with chemotherapy and compared factors associated with associated hearing loss. STUDY DESIGN AND METHODS: This observational cohort study includes a pure-tone audiometry of the patients who underwent chemotherapeutic treatment. Medical history, presence of risk factors for hearing loss, toxic habits, and association with haplogroups have been determined. RESULTS: 40% of patients developed hearing loss after administration of cisplatin, which was bilateral and symmetrical and of high frequencies. The most frequent haplogroup was H with a slight overexpression of groups V and K and a low frequency of groups J and T. No association of the haplogroup types with the hearing loss has been found; however age was revealed as an important determining factor. CONCLUSIONS: Ototoxicity caused by cisplatin is manifested as bilateral, symmetrical, and predominantly high frequency hearing loss. Although we did not find a strong correlation of haplogroups with ototoxicity, our results revealed the existence of a risk group of elderly patients over 60, which are more susceptible to hearing loss induced by cisplatin, than young adults, regardless of preexisting hearing loss.

The cancer stem-cell signaling network and resistance to therapy.

The study of cancer stem cells (CSCs) has shown that tumors are driven by a subpopulation of self-renewing CSCs that retain the capacity to engender the various differentiated cell populations that form tumors. The characterization of CSCs has indicated that CSCs are remarkably resistant to conventional radio- and chemo-therapy. Clinically, the remaining populations of CSC are responsible for metastasis and recurrence in patients with cancer, which can lead to the disease becoming chronic and incurable. Therefore, the elimination of CSCs is an important goal of cancer treatments. Furthermore, CSCs are subject to strong regulation by the surrounding microenvironment, which also impacts tumor responses. In this review, we discuss the mechanisms by which pathways that are defective in CSCs influence ultimately therapeutic and clinical outcomes.

Schwannomas, benign tumors with a senescent phenotype.

BACKGROUND: Schwannomas are benign nerve sheath tumors that only very rarely undergo malignant changes. Oncogenic-induced senescence is a defense mechanism against such malignant transformation. Different molecular pathways are involved in this process, such as RAS-RAF-MAPK. Based on the fact that the RAS-RAF-MAPK pathway is known to be activated in peripheral nerve sheath tumors, this study analyzes senescence markers in Schwannomas to demonstrate the possible role of senescence in their genesis. METHODS: A retrospective immunohistochemical study was done in 39 schwannoma and 18 malignant peripheral nerve sheath tumors (MPNST). Staining for p16INK4a, Ki67, p53 and CyclinD1 was performed in all the cases. Additionally, ß-galactosidase staining was done in those cases in which frozen tissue was available (n=8). RESULTS: Higher levels of p16INK4a (p=0.0001) and lower levels of Ki67 (p=0.0001) were found in Schwannomas. Beta-galactosidase activity was positive in 5/5 Schwannomas and negative in 3/3 MPNST. CONCLUSIONS: Our results support the senescence nature of Schwannomas and the absence of a senescence phenotype in MPNST.

Ribosomal proteins as novel players in tumorigenesis.

Ribosome biogenesis is the most demanding energetic and metabolic expenditure of the cell. The nucleolus, a nuclear compartment, coordinates rRNA transcription, maturation, and assembly into ribosome subunits. The transcription process is highly coordinated with ribosome biogenesis. In this context, ribosomal proteins (RPs) play a crucial role. In the last decade, an increasing number of studies have associated RPs with extraribosomal functions related to proliferation. Importantly, the expression of RPs appears to be deregulated in several human disorders due, at least in part, to genetic mutations. Although the deregulation of RPs in human malignancies is commonly observed, a more complex mechanism is believed to be involved, favoring the tumorigenic process, its progression and metastasis. This review explores the roles of the most frequently mutated oncogenes and tumor suppressor genes in human cancer that modulate ribosome biogenesis, including their interaction with RPs. In this regard, we propose a new focus for novel therapies.

MicroRNAs and cancer stem cells: therapeutic approaches and future perspectives.

Cancer stem cells (CSCs) are a sub-population of cancer cells that possess characteristics associated with normal stem cells but with the peculiarity that they are tumourigenic. This property allows them to persist in the tumour population, causing relapse and metastasis by giving rise to new tumours. Accordingly, if the CSCs were eliminated, then the tumour would simply regress due to differentiation and cell death. By selectively targeting CSCs, it may be possible to treat patients with aggressive, non-resectable tumours and prevent the tumour from metastasising. MicroRNAs are involved in all biological processes, and several studies have demonstrated their function in human tumourigenesis. Importantly, microRNAs have been implicated in the regulation of stem cells and CSCs. The most important concept to emerge with regard to CSC therapy is still controversial because a number of signalling pathways unique to normal stem cells may also be operating in CSCs, and these offer new targets for therapy. This article reviews how the modulation of microRNAs may revert tumour proliferation in vivo and in vitro and how this approach could be transferred to the clinic. Although the delivery of therapeutic microRNAs to target cells is a challenge that still needs to be overcome, microRNAs offer the advantage that they are small molecules that can be easily transported by body fluids, which makes them good candidates for cancer therapy.

p16(Ink4a) overexpression in cancer: a tumor suppressor gene associated with senescence and high-grade tumors.

p16(Ink4a) is a protein involved in regulation of the cell cycle. Currently, p16(Ink4a) is considered a tumor suppressor protein because of its physiological role and downregulated expression in a large number of tumors. Intriguingly, overexpression of p16(Ink4a) has also been described in several tumors. This review attempts to elucidate when and why p16(Ink4a) overexpression occurs, and to suggest possible implications of p16(Ink4a) in the diagnosis, prognosis and treatment of cancer.

Multiple microRNAs rescue from Ras-induced senescence by inhibiting p21(Waf1/Cip1).

Overexpression of Ras(G12V) in primary cells induces a permanent growth arrest called oncogene-induced senescence (OIS) that serves as a fail-safe mechanism against malignant transformation. We have performed a genome-wide small interfering RNA (siRNA) screen and a microRNA (miRNA) screen to identify mediators of OIS and show that siRNA-mediated knockdown of p21(Waf1/Cip1) rescues from Ras(G12V)-induced senescence in human mammary epithelial cells (HMECs). Moreover, we isolated a total of 28 miRNAs that prevented Ras(G12V)-induced growth arrest, among which all of the miR-106b family members were present. In addition, we obtained a number of hits, miR-130b, miR-302a, miR-302b, miR302c, miR-302d, miR-512-3p and miR-515-3p with seed sequences very similar to miR-106b family members. We show that overexpression of all these miRNAs rescues HMECs from Ras(G12V)-induced senescence by prevention of Ras(G12V)-induced upregulation of p21(Waf1/Cip1). Our results establish an important role for the cell cycle inhibitor p21(Waf1/Cip1) in growth control of HMECs and extend the repertoire of miRNAs that modulate the activity of this tumour suppressor.

A new generation of proto-oncogenes: cold-inducible RNA binding proteins.

This review focuses on the roles of two major cold-inducible RNA binding proteins known in human cells: CIRP and RBM3. Both proteins were discovered when they were shown to be induced after exposure to a moderate cold-shock and other cellular stresses such as UV radiation and hypoxia. Initially, it was suggested that these proteins have a suppressive rather stimulatory effect on proliferation; however, proliferative and/or proto-oncogenic functions have recently been assigned to CIRP and RBM3. In a high throughput genetic screen, we recently identified CIRP as an immortalized gene in murine primary cells. On the other hand, the role of RBM3 in transformation has already been demonstrated. Interestingly, both CIRP and RBM3 have been found to be up-regulated in human tumors. This article highlights the roles of CIRP and RBM3 in tumorigenesis, and proposes a model by which CIRP might contribute to senescence bypass by counteracting the deleterious effects of oxidative damage.

Rplp1 bypasses replicative senescence and contributes to transformation.

To determine whether genes expressed by embryonic stem cells have a proliferative effect in primary cells, primary mouse embryonic fibroblasts were infected with an ES cell cDNA library. This led to identification of the ribosomal protein, Rplp1, a member of the P group of ribosomal proteins, whose putative role for bypassing replicative senescence in MEFs was investigated. Our results show that Rplp1 produces a two-fold increase in the expression of an E2F1 promoter and upregulation of cyclin E in MEFs. Therefore, this study is the first to show that overexpression of a single ribosomal protein, Rplp1, is a cause and not a consequence of cell proliferation. In addition, co-expression of Rplp1 with mutant rasVal12 contributed to transformation in NIH3T3 cells, as was evidenced by colony production in soft-agar assays. Moreover, the Rplp1 protein was upregulated in MEFs and NIH3T3 cells upon expression of a p53 dominant negative mutant gene designated p53R175H. Hence, mutation of p53 may facilitate immortalization in vitro by upregulating Rplp1. Lastly, Rplp1 mRNA was found to be upregulated in 16 of 26 human colon cancer biopsy specimens, a finding that may be of relevance to cancer research.

S-adenosylhomocysteine hydrolase downregulation contributes to tumorigenesis.

With the idea to discover novel genes involved in proliferation, we have performed a genome-wide loss-of-function genetic screen to identify additional putative tumor suppressor genes. We have previously identified five genes belonging to different biochemical families. In this report, we focused on the study of one of these genes designated S-adenosylhomocysteine hydrolase (SAHH), which has also been previously identified in an independent short hairpin RNA screening. SAHH inactivation confers resistance to both p53 and p16(INK4)-induced proliferation arrest. Interestingly, SAHH inactivation inhibits p53 transcriptional activity and impairs DNA damage-induced transcription of p21(Cip1). Given that SAHH downregulation modulates senescence in primary cells, we also studied SAHH expression in human tumors at the messenger RNA (mRNA) and protein levels. SAHH mRNA was lost in 50% of tumor tissues from 206 patients with different kinds of tumors in comparison with normal tissue counterparts. Moreover, SAHH protein was also affected in some colon cancers. Such findings may be of relevance to cancer research, suggesting that SAHH might be a largely unexplored tumor suppressor.

Protection from oxidative stress by enhanced glycolysis; a possible mechanism of cellular immortalization.

Reactive oxygen species (ROS) play a crucial role not only in the physiological signal transduction but also in the pathogenesis of several human diseases such as atherosclerosis, neuro-degenerative diseases, metabolic disorders, aging or cancer amongst others. Oxidative stress is also responsible for cellular and organism senescence, in accordance with what Harman initially proposed in the free radical theory of aging. Recent findings support the notion that protection from oxidative stress can increase life span significantly. We reported that enhanced glycolysis could modulate cellular life span with reduction of oxidative stress. Moreover, the tumor suppressor gene p53 controls post-transcriptionally the level of the glycolytic enzyme, phosphoglycerate mutase (PGM). As enhanced glycolysis is a distinctive and prominent feature of cancer cells (termed the Warburg effect), our findings disclosed a novel aspect of the Warburg effect: the connection between senescence and oxidative stress.

New p53 related genes in human tumors: significant downregulation in colon and lung carcinomas.

Human epithelial tumors need to accumulate multiple genetic alterations to form invasive carcinomas. These genetic alterations are related with growth factor receptors, cell signalling, the cell cycle and cell invasiveness. Importantly, cells need to avoid senescence and become immortalized for this process. Recently, five genes: RPS6KA6, HDAC4, KIAA0828, TCP1 and Tip60, which modulate p53-dependent function and avoid senescence were identified in a large-scale RNA interference screen. Twenty colon, 20 prostate and 20 lung carcinomas were studied to investigate whether these genes might be related with human tumors. RNA was extracted from both normal and tumor tissue from each patient. Real-time RT-PCR was performed using TaqMan probes corresponding to the RPS6KA6, HDAC4, KIAA0828, TCP1, Tip60 and p53 genes. In colon carcinomas, the RPS6KA6, HDAC4, KIAA0828 and Tip60 genes were downregulated in tumor tissue as compared with normal tissue (P < 0.001 for all genes). In lung carcinomas, HDAC4, KIAA0820 and Tip60 were downregulated (P < 0.01, P < 0.001 and P < 0.001 respectively). Whereas no significant differences were observed in prostate carcinomas, striking downregulation of the RPS6KA6 and KIAA0828 genes was observed in colon carcinomas and KIAA0828 in a subset of lung carcinomas. mRNA expression of these genes may control p53 function as well as the ras-MAPK pathway, methylation and transcriptional cellular programs. These results could unravel a novel set of regulatory suppressor genes involved in human colon and lung tumors.

Adenovirus E1A orchestrates the urokinase-plasminogen activator system and upregulates PAI-2 expression, supporting a tumor suppressor effect.

Invasiveness and metastatic potential are the two most important properties defining malignancy. The adeno-virus E1A (Ad-E1A) gene has a dual effect as a proliferative gene and as a tumor-suppressor gene, decreasing tumor growth and the metastatic potential of malignant cells. In order to study genes related with the antimetastatic effect of Ad-E1A in human cells, we performed a microarray analysis using OncoChiptrade mark. In three independent experiments, NIH3T3, IMR90 and MDA MB 435 cells were infected with pLPC retroviruses carrying the adenovirus 12S E1A gene or the GFP gene. We analyzed cDNA expression by using the CNIO OncoChipTM, a cDNA microarray containing a total of 6386 genes represented by 7237 clones. uPA, uPAr, tPA, PAI-1 and PAI-2 were also studied at RNA and protein levels. Microarrays of cDNA expression, RT-PCR and Western blot performed in IMR90 E1A-expressing cells showed downregulation of uPA, uPAr, tPA, PAI-1 and upregulation of PAI-2. These results were confirmed in NIH3T3 and MDA MB 435 breast carcinoma cells, with PAI-2 upregulation by RT-PCR and Western blot. In addition, zymographic analysis demonstrated that E1A expression greatly reduced the gelatinase activity of the pro-MMP2 and -MMP9 proteins. We propose that adenovirus E1A may orchestrate the expression of most members of the urokinase-plasminogen activation system, downregulating potentially invasive genes and upregulating PAI-2, which is associated with a better prognosis in human tumors.

HRAS1 minisatellite alleles in colorectal carcinoma: relationship to microsatelite instability.

OBJECTIVES: To further evaluate sporadic colon carcinoma risk associated with rare HRAS1 VNTR alleles, the relationship with microsatellite instability and with HRAS1 VNTR instability. MATERIALS AND METHODS: The HRAS1 VNTR was genotyped in 121 tumors and normal samples from sporadic colon carcinoma patients (47 right and 74 left colon) and in 109 samples from healthy individuals. The HRAS1 alleles were identified using PCR and automatic fluorescent electrophoresis detection combined with MVR-PCR (Minisatellite Variant Repeat-Polymerase Chain Reaction). Microsatellite Instability (MI) was analysed with 10 microsatellite markers. RESULTS: A relative risk of 3.04 (95% CI: 1.16-4.92) associated with rare alleles was obtained. No HRAS1 minisatellite instability was present in the tumors. Samples with MI were equally distributed between the common and rare HRAS1 allele groups, while the distribution of HRAS1 alleles in samples with MI was similar in right and left colorectal carcinoma. CONCLUSION: Rare HRAS1 VNTR alleles are associated with colorectal carcinoma risk independent of the tumor location. MI is not likely to be involved in the same underlying defect that generates rare HRAS1 alleles in colorectal carcinoma.

Tumor heterogeneity: morphological, molecular and clinical implications.

Malignant tumors are characterized by their great heterogeneity and variability. There are hundreds of different types of malignant tumors that harbour many oncogenic alterations. The tumor heterogeneity has important morphological, molecular and clinical implications. Except for some hematopoietic and lymphoproliferative processes and small cell infant tumors, there are not specific molecular alterations for most human tumors. In this review we summarize the most important aspects of carcinogenesis and chemoradiosensitivity of malignant cells. In this regard, some oncogenes such as neu, ras and bcl-2 have been associated with cellular resistance to treatment with anticancer agents. The knowledge of oncogenic alterations involved in each tumor can be important to correlate the morphological features, the genetic background, the prognosis and the clinical response to treatment with anticancer agents. Based on the molecular background of the tumor there are new cancer gene therapy protocols. For example using adenovirus Ela in tumors with overexpression of neu oncogene, inhibitors of tyrosine kinase specific for the PDGF receptor in glioma, inhibitors of farnesil transferase to prevent ras activity in tumors with mutations in the ras gene.

Microsatellite instability and p53 mutations in sporadic right and left colon carcinoma: different clinical and molecular implications.

BACKGROUND: Left and right colon carcinomas can display different clinical, pathologic, and genetic characteristics. The purpose of this study was to characterize multiple molecular genetic alterations in sporadic colon carcinoma and to correlate them with the location of the tumors and with lymph node metastasis. METHODS: One hundred and twenty-five cases of sporadic colon carcinoma (50 in the right colon and 75 in the left colon in patients with no family history of colon carcinoma) were studied. Status of the p53 gene was assessed by polymerase chain reaction (PCR), single strand conformation polymorphism, and sequencing at exons 5-8. Microsatellite instability was analyzed with five microsatellite markers at chromosome 18. The mismatch repair genes hMLH1 and hMSH2 were studied in tumors found to have microsatellite instability by PCR and sequencing. RESULTS: In the 125 cases studied, there were 53 tumors with mutations of the p53 gene. p53 mutations correlated with lymph node metastases from right colon carcinoma cases (61%), and all cases with p53 mutations and microsatellite instability were AJCC/UICC Stage III (Dukes Stage C). In the right colon carcinoma cases the rate of microsatellite instability was related to the tumor size (19% in tumors measuring < 4 cm, and 34% in tumors measuring > 4 cm). No correlation between microsatellite instability and p53 mutations was detected. In the left colon carcinoma cases, p53 mutations were detected in 41% of tumors and microsatellite instability in 14%; neither finding was related to the tumor size. Mutations of the hMLH1 and hMSH2 mismatch repair genes were detected in 7 of 24 cases with marked microsatellite instability. CONCLUSIONS: Microsatellite instability is prone to occur in sporadic right colon carcinoma during tumor growth and is not associated significantly with mutations in the hMLH1 and hMSH2 mismatch repair genes or in the p53 gene. Concomitant detection of microsatellite instability and p53 mutations in right colon carcinoma is associated with the presence of lymph node metastases.