Use of the EM algorithm to detect QTL affecting multiple-traits in an across half-sib family analysis

QTL detection experiments in livestock species commonly use the half-sib design. Each male is mated to a number of females, each female producing a limited number of progeny. Analysis consists of attempting to detect associations between phenotype and genotype measured on the progeny. When family sizes are limiting experimenters may wish to incorporate as much information as possible into a single analysis. However, combining information across sires is problematic because of incomplete linkage disequilibrium between the markers and the QTL in the population. This study describes formulæ for obtaining MLEs via the expectation maximization (EM) algorithm for use in a multiple-trait, multiple-family analysis. A model specifying a QTL with only two alleles, and a common within sire error variance is assumed. Compared to single-family analyses, power can be improved up to fourfold with multi-family analyses. The accuracy and precision of QTL location estimates are also substantially improved. With small family sizes, the multi-family, multi-trait analyses reduce substantially, but not totally remove, biases in QTL effect estimates. In situations where multiple QTL alleles are segregating the multi-family analysis will average out the effects of the different QTL alleles.     

Oncogenic BRAF induces chronic ER stress condition resulting in increased basal autophagy and apoptotic resistance of cutaneous melanoma

The notorious unresponsiveness of metastatic cutaneous melanoma to current treatment strategies coupled with its increasing incidence constitutes a serious worldwide clinical problem. Moreover, despite recent advances in targeted therapies for patients with BRAF^V600E mutant melanomas, acquired resistance remains a limiting factor and hence emphasises the acute need for comprehensive pre-clinical studies to increase the biological understanding of such tumours in order to develop novel effective and longlasting therapeutic strategies. Autophagy and ER stress both have a role in melanoma development/progression and chemoresistance although their real impact is still unclear. Here, we show that BRAF^V600E induces a chronic ER stress status directly increasing basal cell autophagy. BRAF^V600E-mediated p38 activation stimulates both the IRE1/ASK1/JNK and TRB3 pathways. Bcl-XL/Bcl-2 phosphorylation by active JNK releases Beclin1 whereas TRB3 inhibits the Akt/mTor axes, together resulting in an increase in basal autophagy. Furthermore, we demonstrate chemical chaperones relieve the BRAF^V600E-mediated chronic ER stress status, consequently reducing basal autophagic activity and increasing the sensitivity of melanoma cells to apoptosis. Taken together, these results suggest enhanced basal autophagy, typically observed in BRAF^V600E melanomas, is a consequence of a chronic ER stress status, which ultimately results in the chemoresistance of such tumours. Targeted therapies that attenuate ER stress may therefore represent a novel and more effective therapeutic strategy for BRAF mutant melanoma.Cutaneous melanoma represents one of the most aggressive and difficult to treat forms of human cancer, with a worldwide incidence that has steadily increased over the past 40 years.^{1, 2}Notoriously unresponsive to conventional chemotherapy, metastatic disease is highly invasive and evolves with an extensive repertoire of molecular defences against immunological and cytotoxic attack.³Although linked to exposure to ultraviolet light, it is widely accepted that both genotypic and phenotypic changes in melanocytes predispose to melanocyte transformation and the onset of melanoma.^{4, 5}Surprisingly, p53 mutations are very rare in melanoma, but activity is, however, impaired through direct or indirect inactivation of key elements of this pathway, including through the suppression of APAF-1 expression,⁶ loss of PTEN function,⁷ dysregulation of Bcl-2 expression,⁸ upregulation of the anti-apoptotic protein Mcl-1 together with its altered slice variant expression ^{9, 10} and the ER chaperone GRP78.^{11, 12, 13} Oncogenic mutations, however, in the Ras/Raf pathway are the most well-described genetic mutations associated with melanoma development and progression.¹⁴ Indeed, up to 90% of all melanomas harbour activating NRAS or BRAF mutations, with BRAF^V600E representing more than 90% of BRAF mutations,^{15, 16} the consequence of which is the constitutive activation of RAF-extracellular signal-regulated kinase/ERK signalling promoting melanoma proliferation and resistance to apoptosis.¹⁷ Nevertheless, mutation of NRAS/BRAF alone is not sufficient to initiate melanomagenesis, because these common mutations are also present in benign nevi, thereby highlighting the requirement of other factors to drive melanocyte transformation and melanoma development.^{15, 16} Dysregulation of autophagy has accordingly been postulated as a secondary event contributing to melanoma progression and, importantly, also has a key role in chemoresistance.^{18, 19, 20}Autophagy is the principal catabolic process for the bulk degradation and recycling of aged/damaged cellular components, organelles and proteins through the formation of a double-membraned cytosolic vesicle able to wrap targeted material. The subsequent fusion with lysosomes and degradation of cargo provide nutrients in times of environmental stress, such as nutrient deprivation or hypoxia.²¹ Though essential for the maintenance of cellular homeostasis under conditions of nutrient deprivation, paradoxically, autophagy promotes both tumour suppression and tumour development.²² Although the accumulation of damaged organelles/cytosolic proteins may lead to cellular transformation, autophagy may also sustain tumour growth in a microenvironment which is commonly poor of oxygen and nutrients.²² Thus, not surprisingly, autophagy activation is frequently observed in late-stage malignancy although the molecular mechanisms mediating its activation/regain of function remain unclear.ER stress may also constitute a key secondary event in melanoma development.²³ Primarily a cytoprotective pro-survival process, ER stress is activated as a result of accumulated unfolded proteins, protein overload or depletion of ER calcium stores and mediated through the activity of the master ER chaperone Grp78 and three signalling pathways; PERK/eIF2α/ATF4, IRE-1/Xbp-1 and ATF6 which collectively maintain ER homeostasis through the instigation of an unfolded protein response (UPR)²⁴ or sustained ER stress may lead to the induction of apoptosis.^{25, 26} Increasing evidence indicates that nutrient deprivation and hypoxia lead to activation of the UPR in various solid tumours, frequently correlating with resistance to chemotherapy.²⁷ The accepted hypothesis is thus that activation of the UPR in cancer cells enables their adaption to such ER stress resulting in the resistance to apoptosis through the persistent expression of pro-survival instead of pro-apoptotic proteins.²⁸Although under stress conditions, autophagy and ER stress seem to act in parallel, indeed they are closely related, because one can regulate the other and vice versa. In fact, ER stress is able to promptly stimulate autophagy,²⁶ whereas autophagy selectively removes the membranes of the endoplasmic reticulum at the end of the UPR, although the molecular mechanisms are still largely unclear.²⁹In the present study, we investigated the link between oncogenic BRAF^V600E and increased basal autophagy in melanoma cells, highlighting the pivotal role played by ER stress, possibly responsible for tumour growth and chemoresistance.     

Expression of collier in the premandibular segment of myriapods: support for the traditional Atelocerata concept or a case of convergence?

Background  

A recent study on expression and function of the ortholog of the Drosophila collier (col) gene in various arthropods including insects, crustaceans and chelicerates suggested a de novo function of col in the development of the appendage-less intercalary segment of insects. However, this assumption was made on the background of the now widely-accepted Pancrustacea hypothesis that hexapods represent an in-group of the crustaceans. It was therefore assumed that the expression of col in myriapods would reflect the ancestral state like in crustaceans and chelicerates, i.e. absence from the premandibular/intercalary segment and hence no function in its formation.     

The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. An almost identical SMN2 gene is unable to compensate for this deficiency because a single C‐to‐T transition at position +6 in exon‐7 causes skipping of the exon by a mechanism not yet fully elucidated. We observed that the C‐to‐T transition in SMN2 creates a putative binding site for the RNA‐binding protein Sam68. RNA pull‐down assays and UV‐crosslink experiments showed that Sam68 binds to this sequence. In vivo splicing assays showed that Sam68 triggers SMN2 exon‐7 skipping. Moreover, mutations in the Sam68‐binding site of SMN2 or in the RNA‐binding domain of Sam68 completely abrogated its effect on exon‐7 skipping. Retroviral infection of dominant‐negative mutants of Sam68 that interfere with its RNA‐binding activity, or with its binding to the splicing repressor hnRNP A1, enhanced exon‐7 inclusion in endogenous SMN2 and rescued SMN protein expression in fibroblasts of SMA patients. Our results thus indicate that Sam68 is a novel crucial regulator of SMN2 splicing.     

IGFBP7 induces apoptosis of acute myeloid leukemia cells and synergizes with chemotherapy in suppression of leukemia cell survival

Despite high remission rates after chemotherapy, only 30–40% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis. This extremely poor prognosis of AML is mainly caused by treatment failure due to chemotherapy resistance. Chemotherapy resistance can be caused by various features including activation of alternative signaling pathways, evasion of cell death or activation of receptor tyrosine kinases such as the insulin growth factor-1 receptor (IGF-1R). Here we have studied the role of the insulin-like growth factor-binding protein-7 (IGFBP7), a tumor suppressor and part of the IGF-1R axis, in AML. We report that IGFBP7 sensitizes AML cells to chemotherapy-induced cell death. Moreover, overexpression of IGFBP7 as well as addition of recombinant human IGFBP7 is able to reduce the survival of AML cells by the induction of a G2 cell cycle arrest and apoptosis. This effect is mainly independent from IGF-1R activation, activated Akt and activated Erk. Importantly, AML patients with high IGFBP7 expression have a better outcome than patients with low IGFBP7 expression, indicating a positive role for IGFBP7 in treatment and outcome of AML. Together, this suggests that the combination of IGFBP7 and chemotherapy might potentially overcome conventional AML drug resistance and thus might improve AML patient survival.Only 30–40% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis.¹ This extremely poor prognosis is mainly caused by treatment failure due to chemotherapy resistance. This resistance is often a multifactorial phenomenon that can include enhanced expression or activation of receptor tyrosine kinases such as the insulin growth factor-1 receptor (IGF-1R).^{2, 3} The IGF-1R stimulates proliferation, protects cells from apoptosis and has been implicated in the development and maintenance of various cancers.^{4, 5} Several oncogenes require an intact IGF-1R pathway for their transforming activity⁶ and moreover, disruption or inhibition of IGF-1R activity has been shown to inhibit the growth and motility of a wide range of cancer cells in vitro and in mouse models.^{4, 5} IGF-1Rs are membrane receptors and binding of their ligand, the insulin-like growth factor-1 (IGF-1), results in receptor phosphorylation and activation of MAPK and PI3K/Akt signaling.⁴ Importantly, IGF-1, normally produced by the liver and bone marrow stromal cells, can stimulate the proliferation of cancer cells in vitro and genetic manipulations that reduce IGF-1 signaling can lead to decreased tumor growth.^{7, 8}In hematological malignancies, a role for IGF-1 signaling has been demonstrated in multiple myeloma (MM) where it stimulates growth and potently mediates survival.⁹ Several anti-IGF-1R strategies have been shown to inhibit MM growth.^{10, 11} In AML, expression of the IGF-1R and IGF-1 was detected in AML cell lines and primary AML blasts and stimulation with IGF-1 can promote the growth of AML cells.^{12, 13, 14} In addition, neutralizing IGF-1R antibodies and the tyrosine kinase inhibitors (TKIs) NVP-AEW541 and NVP-ADW742, have been shown to inhibit proliferation and to induce apoptosis.^{15, 16}In addition to its mitogenic and anti-apoptotic roles, directly influencing tumor development, IGF-1R appears to be a critical determinant of response to numerous anti-cancer therapies, including TKIs and chemotherapy.^{2, 3, 17, 18, 19, 20, 21, 22} In AML, activated IGF-1R signaling has been linked to cytarabine resistance, a drug included in every AML treatment schedule.¹⁷ Notably, in several cancer cell lines, a small subpopulation of drug-tolerant cancer cells exists that maintains their viability, after treatment with a lethal drug dose, via engagement of the IGF-1R.¹⁸The activity of the IGF-1R is tightly controlled at multiple levels, including their processing, endocytosis, trafficking and availability of its ligands.⁴ Ligand bioavailability is partly controlled by the family of secreted insulin-like growth factor-binding protein (IGFBP1 to IGFBP6), which can bind to IGFs therewith regulating the interaction of these ligands to their receptors. However, as IGFBPs are able to induce IGF-dependent and IGF-independent effects, the results of several studies on their role in cancer cell survival appeared to be controversial and complex.^{23, 24} In addition to IGFBPs, various IGFBP-related proteins have been identified.^{23, 25} One of these is the IGFB-related protein 1, also known as insulin-like growth factor-binding protein-7 (IGFBP7). IGFBP7 has 30% homology to IGFBP1 to IGFBP6 in its N-terminal domain and functions predominantly as a tumor suppressor.^{23, 24, 25, 26} In contrast to IGFBP1 to IGFBP6, which bind to the IGFs,²³ IGFBP7 is a secreted protein that can directly bind to the IGF-1R and thereby inhibits its activity.²⁷ The abundance of IGFBP7 is inversely correlated with tumor progression in hepatocellular carcinoma.²⁸ Importantly, decreased expression of IGFBP7 has been associated with therapy resistance^{29, 30} and increasing IGFBP7 levels can inhibit melanoma and breast cancer growth.^{31, 32} IGFBP7 was originally identified as being involved in Raf-mediated apoptosis and senescence³³ and also has been shown to induce senescence in mesenchymal stromal cells.³⁴We established that IGFBP7 induces a cell cycle block and apoptosis in AML cells and cooperates with chemotherapy in the induction of leukemia cell death. AML patients with low IGFBP7 expression have a worse outcome than patients with high IGFBP7 expression, indicating that AML patients might benefit from a combination therapy consisting of chemotherapy and IGFBP7. Our results define IGFBP7 as a focus to enhance chemotherapy efficacy and improve AML patient survival.