Alterations in the ribosomal machinery in cancer and hematologic disorders

Diamond-blackfan anemia (DBA):DBA is a congenital aregenerative anemia accompanied by erythroblastopenia (less than 5% of nucleated cells in the bone marrow are erythroblasts). It is often discovered before the age of two years. There are associated developmental malformations in 40% of the cases. The malformations most often involve the head and neck (palatine cleft, ogival palate, webbed appearance of the neck, microphthalmia, absent lower eyelashes, deformed external ears, micrognathia), limbs (triphalangeal thumb, supernumerary digits, syndactyly), urogenital tract, heart and/or the spinal cord.In 1999, Draptchinskaia reported mutations in the gene encoding ribosomal protein S19 in patients with Diamond-Blackfan anemia [4]. Since then, mutations in a number of ribosomal proteins have been identified in 50% of DBA patients. Mutations in RPS19 and RPS 24, which were the first two mutations identified, were shown to impair the pre-RNA processing of 18 S RNA which in turn lead to reduced production of 40 S ribosomal subunit and mature 80 S ribosomes [5‐7]. The other ribosomal protein gene mutations found in DBA involve RPS7, RPS15, RPS17, RPS27A, RPL5, RPL11, RPL35A and RPL36 [8‐10]. Current therapy for DBA includes steroids and chronic blood transfusions, with the only definitive therapy being bone marrow transplantation [11].

5q- syndrome:5q- syndrome is a disorder resulting from deletion of the long arm of chromosome 5 and is characterized by macrocytic anemia with normal/elevated platelets with hypolobulated micromegakaryocytes. It is found predominantly in females of advanced age. It is now classified as an independent subtype of MDS, with better prognosis and a relatively low rate (10%) of progression to acute myeloid leukemia compared with other subtypes of MDS [12]. In patients with 5q- syndrome, 1 allele of RPS14 is deleted and haploinsufficient expression of RPS14 has been found in patients [5, 13, 14]. Decreased expression of RPS14 causes ineffective erythropoiesis. It has also been shown that reexpression of RPS14 in samples from patients with 5q- syndrome rescues erythropoiesis. As discussed earlier, RPS14 deficiency causes impaired processing of 18 S RNA and in turn leads to reduced levels of the 40 S subunit [15]. The heterozygous deletions of chromosome 5q are large and haploinsufficiency of other genes may also contribute to the 5q- phenotype [16]. For example, haploinsufficiency of micrornas, miR-145 and miR-146a, may be responsible for thrombocytosis seen in this disorder [17, 18]. However, the erythroid defect, which is the aspect of the phenotype most analogous to DBA, has been shown both in in-vivo (murine) and in-vitro models to be a result of haploinsufficiency of RPS14 [15, 19]. Lenalidomide has been shown to be very effective in the treatment of patients with 5q- syndrome. In a phase 2 trial in low risk MDS patients with 5q deletions, lenalidomide decreased transfusion requirements in 76% and 61% had a complete cytogenetic response [20, 21].

Treacher collins syndrome (TCS):Also called mandibulofacial dysostosis, TCS is a rare autosomal dominant congenital disorder characterized by craniofacial abnormalities such as micrognathia, malformed or absent external ears, underdeveloped zygoma, downward slanting eyes, conductive hearing loss, colobomata of the lower eye lids, cleft palate, brachycephaly, variable degree of facial nerve involvement [22]. Intelligence of patients with TCS is usually normal but psychosocial problems associated with the facial deformity affect the quality of life of a number of patients. In 1996, TCOF1 gene mutation was found to be the cause for TCS. TCOF1 encodes a protein called Treacle, which is a constituent of one of the preribosomal ribonucleoprotein complexes [23]. It has been shown to be essential for the transcription of ribosomal DNA and it may also be involved in the methylation of rRNA [24]. Mice haploinsufficient for TCOF1 have diminished production of ribosomes and this has been shown to correlate with decreased production of neural crest precursor cells [25]. This in turn leads to decreased number of neural crest cells migrating to the first and second pharyngeal arches, resulting in the development of the syndrome. An interesting study by Jones et al. showed that chemical and genetic inhibition of p53 in these mice could prevent the development of craniofacial abnormalities [26]. The link between ribosomopathies and p53 activation will be discussed later in the review.

Cartilage hair hypoplasia:CHH is an autosomal recessive disorder that results in short limb dwarfism [27]. It is associated with T cell and B cell immunodeficiency. Hematologic abnormalities can also include macrocytic anemia, apart from lymphopenia. Management is supportive, with stem cell transplantation being the only curative option. It is indicated specially in patients with severe T cell deficiency, although transplantation does not cure skeletal abnormalities [28]. The genetic defect in cartilage hair hypoplasia has been identified as a mutation in the gene for RMRP, mapped to 9p12 [29]. RMRP is a ribonucleoprotein present in the nucleus and mitochondria. RMRP is involved in the cleavage of RNA in mitochondrial DNA synthesis and cleavage of pre-ribosomal RNA (pre-RNA) [30]. RMRP is required for cell growth, consistent with observations that a generalized defect in cell growth is seen in T cells, B cells and fibroblasts. In 2005, a study reported that CHH cells with RMRP mutation were found to have increased levels of cyclin B2 mRNA [31]. Cyclin B2 is known to contribute to chromosomal instability through alterations of the mitotic spindle checkpoint, which suggests another explanation for the bone marrow dysfunction [32].

Shwachman diamond syndrome (SDS):SDS is a rare autosomal recessive disorder characterized by exocrine pancreatic insufficiency, bone marrow dysfunction, skeletal abnormalities and a predisposition to leukemia [33]. It is the third most common inherited bone marrow failure syndrome after Fanconi anemia and Diamond Blackfan anemia. Patients typically present in early infancy with malabsorption, steatorrhea, failure to thrive and deficiency of Vitamins A, D, E and K. Neutropenia is the most common hematologic abnormality seen in patients. Data from a large international cohort study consisting of 88 patients showed neutropenia in 98% of the patients, anemia in 42%, thrombocytopenia in 34% and pancytopenia in 19%. In 2003, Boocock et al. reported causal mutations in the SBDS gene, named after Shwachman-Bodian-Diamond, who first reported the syndrome. About 90% of the patients were found to have biallelic mutations of the SBDS gene [34]. Although the precise function of the gene is unclear, there is evidence suggesting a role in ribosome biogenesis and ribosome processing [35]. SDS cells have been shown to underexpress many ribosomal protein genes including RPS9, RPS20, RPL6, RPL15, RPL22, RPL23 and RPL29 and genes involved in rRNA and mRNA processing [36]. SBDS functions in ribosome synthesis by promoting the recycling of eukaryotic initiation factor 6 (eIF6) in a GTP-dependent manner. Finch et al. stated that a perturbation in this function causes SBDS [37]. However, SBDS is a multifunctional protein and it is also possible that non ribosomal mechanisms have a dominant role in the pathogenesis of the syndrome. Of note is SBDS’ role in stabilizing the mitotic spindle [38], the absence of which may lead to chromosomal instability and therefore bone marrow failure.

Dyskeratosis congenita (DKC):DKC is a rare, progressive bone marrow failure syndrome characterized by the triad of reticulated skin hyperpigmentation, nail dystrophy and oral leukoplakia. Early mortality is often associated with bone marrow failure, infections, fatal pulmonary complications or malignancy [39]. In all cases of DKC, the causative mutations are in the telomerase complex, composed of telomerase reverse transcriptase (TERT), telomerase RNA (TERC) and dyskerin, which adds specific DNA sequences to the ends of chromosomes and counters some of the normal shortening that occurs during DNA replication [40]. In X-linked recessive DKC, which has a more severe phenotype compared to the autosomal dominant DKC, the mutation occurs in the DKC1 gene, which codes for dyskerin [41]. Apart from being a part of the telomerase complex, dyskerin is also involved in rRNA modification (pseudo-uridylation of rRNA). However, the functional consequence of the defect in pseudouridylation of rRNA remains unclear. Patients with the autosomal dominant form of DKC have mutations in TERC; and two families with the autosomal recessive form of DKC have mutations in TERT which suggest that a defect in telomerase activity alone is sufficient for defective hematopoiesis [42]. However, a study on a DKC1 mutant mouse showed pseudouridylation of rRNA before the manifestation of clinical features of DKC whereas the reduction in telomere length became evident only in the later generations [43]. This suggests that in addition to the telomerase complex mutations, defects in rRNA modification might have a contributory role in the clinical manifestations of DKC.

Common variable immunodeficiency (CVID):CVID is a disorder that involves low levels of most or all of the immunoglobulin classes, a lack of B lymphocytes or plasma cells that are capable of producing antibodies and frequent bacterial infections. A diagnosis of CVID is reserved for those with an undefined B cell dysfunction. About 50% of the patients also have T lymphocyte dysfunction. The recognition of a patient with DBA who subsequently developed CVID and the finding of a mutation in the SBDS gene in another patient with CVID has led to the hypothesis that ribosome biogenesis defects are also responsible for a subset of CVID. A review of the literature shows that patients with ribosomal defects may share abnormalities of T and B cell development with many features of CVID, and which may not be recognized as such by non-immunologists [44].

Turner syndrome:Turner syndrome results from a chromosomal abnormality in which all or part of one of the female sex chromosomes is absent. Monosomy X in which an entire X chromosome is missing, is most common. Characteristic physical abnormalities include short stature, webbed neck, broad chest, low hairline, low-set ears and edema. Girls with Turner Syndrome typically experience gonadal dysfunction resulting in amenorrhea and sterility. Other health problems frequently present include congenital heart disease, hypothyroidism, diabetes, vision and hearing problems, autoimmune diseases and developmental delays.Human sex linked genes RPS4X and RPS4Y encode 2 isoforms of ribosomal protein S4. RPS4 is a component of 40 S subunit and haploinsufficiency of RPS4 is hypothesized to lead to some features of Turner’s syndrome such as panhypogammaglobulinemia, low IgM, decreased T and B cell numbers [45, 46].

Protein biosynthesis function of ribosomal proteins in cancer: Both tumor suppressors and oncogenes have been shown to modulate the ribosome protein biosynthesis as well as ribosome translation initiation in various models [59].. MYC, a proto-oncogene product, regulates the mature ribosome biogenesis by modifying the genes of necessary factors involved in ribosomal assembly. Its overexpression in tumor cells increases the expression and activity of ribosomal components. Therefore, regulation of protein synthesis could be an important mechanism by which MYC regulates cell growth and initiates tumorigenesis [60]. PTEN, a tumor suppressor, also regulates the mature ribosome formation, by suppressing the activity of RPS6K [61]. These data suggest that perturbation in the protein biosynthesis fuction of ribosomal proteins may be an important contributor to carcinogenesis.

Interaction of p53 with ribosomal proteins: Likely mechanism involved in some features of ribosomopathies and associations between ribosomal proteins and cancer.A number of events, including haploinsufficiency of some ribosomal proteins, impart instability to ribosomal biogenesis and cause nucleolar stress. In response to this stress, some ribosomal proteins (RPL5, RPL11, RPL23, RPS7 and RPL26) bind to MDM2 and block MDM2-mediated p53 ubiquitination and degradation, resulting in p53-dependent cell cycle arrest. By doing so, the ribosomal proteins play a crucial role in connecting deregulated cell growth with inhibition of cell division. The ribosomal protein-MDM2-p53 signalling pathway provides a molecular switch that may constitute a surveillance network monitoring the integrity of ribosomal biogenesis [62]. (Figure 1) In the Rps19 mutant mouse model used to study DBA, induction of p53 and p53 target genes was identified with the mutation. These mice had decreased hematocrit and increased MCV and phenocopied the human disease. The anemia observed in these mice was partially abrogated with monoallelic inactivation of p53. Homozygous inactivation of p53 in Rps19 mutant mice fully corrected the hematologic abnormality [63]. Furthermore, when the mice with the conditional deletion of a set of genes found in the common deleted region of the 5q- syndrome were crossed with p53 null mice, there was a complete rescue of the erythroid phenotype [19]. These findings indicate that p53 induction is critical for the macrocytic anemia caused by ribosomal dysfunction. P53 activation can also mediate tumor cytotoxicity mediated by reduction in levels of certain ribosome proteins. Decreased levels of RPS9 lead to growth inhibition in osteosarcoma cell lines. It has been shown that depletion of RPS9 provokes a rapid loss of the nucleolar protein pool, impaired production of mature 18 S ribosomal RNA and activation of the p53 tumor suppressor pathway. The combination of a defective ribosome biogenesis pathway and p53 activation results in unexpectedly strong anti-proliferative responses in human tumor cell lines [64]. While decreased expression of some ribosomal proteins can activate p53 as part of a cellular stress response, it is also evident that some other ribosomal proteins have direct and more specific roles in p53 mRNA translation. Haploinsufficiency of a subset of r-proteins (L35, S15a, S8, L36, L7, S7, L13, S29, L23a) in zebrafish is linked to the development of malignant peripheral nerve sheath tumors. Interestingly, these nerve sheath tumors fail to produce p53 protein, despite presence of p53 mRNA [65]. Moreover, RPL26 plays a role in enhancing p53 mRNA translation during the DNA damage response by direct interactions with both MDM2 protein and p53 mRNA [66].Hence, ribosomal proteins have more dynamic roles in regulating the p53 tumor suppressor pathway than was previously thought and this complex interaction appears to be the most significant extraribosomal function of the ribosomal proteins, through which they exert an effect on the cell cycle and apoptosis. It could explain not only some features of ribosomopathies (particularly DBA and 5q- syndrome) but may also have a significant role in carcinogenesis (as elicited in the RPS9 silencing study).

Non p53 mediated extraribosomal functions of ribosomal proteins in cancer:There is some evidence showing that other extraribosomal function of ribosomal proteins may also be directly involved in tumorigenesis. RPs can directly regulate the affect of oncogene and tumor suppressors on DNA replication, transcription and translation. The recombination of human trk proto-oncogene with RPL7a activates its oncogenic function [67]. RPS29, whose mRNAs is much higher in quiescent cells than that in growth phase of endothelial cells, has tumor suppressor activity for ras transformed NIH3T3 cells [68]. On the other hand, the overexpression of RPS3a gene has been shown to promote tumorigenesis in nude mice works via suppression of apoptosis by inducing synthesis of anti-apoptosis proteins [69]. These studies indicate that extraribosomal functions of distinct ribosomal proteins may also regulate tumorigenesis.