Rho-mediated signaling promotes BRAF inhibitor resistance in de-differentiated melanoma cells.

Over half of cutaneous melanoma tumors have BRAFV600E/K mutations. Acquired resistance to BRAF inhibitors (BRAFi) remains a major hurdle in attaining durable therapeutic responses. In this study we demonstrate that ~50-60% of melanoma cell lines with vemurafenib resistance acquired in vitro show activation of RhoA family GTPases. In BRAFi-resistant melanoma cell lines and tumors, activation of RhoA is correlated with decreased expression of melanocyte lineage genes. Using a machine learning approach, we built gene expression-based models to predict drug sensitivity for 265 common anticancer compounds. We then projected these signatures onto the collection of TCGA cutaneous melanoma and found that poorly differentiated tumors were predicted to have increased sensitivity to multiple Rho kinase (ROCK) inhibitors. Two transcriptional effectors downstream of Rho, MRTF and YAP1, are activated in the RhoHigh BRAFi-resistant cell lines, and resistant cells are more sensitive to inhibition of these transcriptional mechanisms. Taken together, these results support the concept of targeting Rho-regulated gene transcription pathways as a promising therapeutic approach to restore sensitivity to BRAFi-resistant tumors or as a combination therapy to prevent the onset of drug resistance.

Autoimmune pancreatitis presenting as a pancreatic mass mimicking malignancy.

Autoimmune pancreatitis is a rare cause of chronic pancreatitis and pancreatic mass. We describe a case of focal autoimmune pancreatitis in a 51-year-old man presenting with obstructive jaundice and pancreatic mass, mimicking malignancy. The immunological test was suggestive of autoimmune pancreatitis, and the patient responded well to a course of steroids, with complete resolution of the pancreatic mass. Autoimmune pancreatitis, therefore, must be kept in mind as a differential diagnosis of pancreatic mass. Recognition of this disease by its typical radiological and serological findings may help to avoid unnecessary surgical resection.

Cholestasis and biliary dilatation associated with chronic ketamine abuse: a case series.

Ketamine is a dissociative anaesthetic agent that is still widely used in veterinary and human medicine. It is increasingly being used as a recreational hallucinogenic drug. Chronic ketamine abuse is known to account for lower urinary tract symptoms and urinary bladder dysfunction. There is now emerging evidence that ketamine misuse is also associated with abnormal liver function tests and biliary tract abnormality. We report three cases of chronic ketamine misuse in three young men who all presented with obstructive jaundice and biliary tract abnormality. We also describe the clinical features, radiological findings and potential underlying mechanisms for this new entity.

Altered transcription in yeast expressing expanded polyglutamine.

Expanded polyglutamine tracts are responsible for at least eight fatal neurodegenerative diseases. In mouse models, proteins with expanded polyglutamine cause transcriptional dysregulation before onset of symptoms, suggesting that this dysregulation may be an early event in polyglutamine pathogenesis. Transcriptional dysregulation and cellular toxicity may be due to interaction between expanded polyglutamine and the histone acetyltransferase CREB-binding protein. To determine whether polyglutamine-mediated transcriptional dysregulation occurs in yeast, we expressed polyglutamine tracts in Saccharomyces cerevisiae. Gene expression profiles were determined for strains expressing either a cytoplasmic or nuclear protein with 23 or 75 glutamines, and these profiles were compared to existing profiles of mutant yeast strains. Transcriptional induction of genes encoding chaperones and heat-shock factors was caused by expression of expanded polyglutamine in either the nucleus or cytoplasm. Transcriptional repression was most prominent in yeast expressing nuclear expanded polyglutamine and was similar to profiles of yeast strains deleted for components of the histone acetyltransferase complex Spt/Ada/Gcn5 acetyltransferase (SAGA). The promoter from one affected gene (PHO84) was repressed by expanded polyglutamine in a reporter gene assay, and this effect was mitigated by the histone deacetylase inhibitor, Trichostatin A. Consistent with an effect on SAGA, nuclear expanded polyglutamine enhanced the toxicity of a deletion in the SAGA component SPT3. Thus, an early component of polyglutamine toxicity, transcriptional dysregulation, is conserved in yeast and is pharmacologically antagonized by a histone deacetylase inhibitor. These results suggest a therapeutic approach for treatment of polyglutamine diseases and provide the potential for yeast-based screens for agents that reverse polyglutamine toxicity.

Cross-cultural validation of the McGill Quality of Life questionnaire in Hong Kong Chinese.

The main focus of palliative care services is to improve patients' quality of life (QOL). The potential value of assessment of QOL in palliative care is being increasingly recognized. The McGill Quality of Life questionnaire (MQOL) is designed specifically for palliative care patients, but its cross-cultural validity needs to be determined before it can be applied in populations of different cultures and ethnic groups. The cross-cultural validity of MQOL was investigated using a translated and modified version in Chinese--the MQOL-HK--in 462 palliative care patients in Hong Kong. Results show that the MQOL-HK is acceptable, valid and reliable. There is good acceptability, construct validity, convergent and divergent validity, test-retest and inter-rater reliability. Our study confirms that QOL does have cross-culturally robust constructs. Principal components analysis shows that the domains of physical, psychological, existential and support are all relevant and applicable in Chinese culture. Multiple regression analysis reveals that existential domain is the most important domain in predicting overall QOL. 'Face', eating and sex are additional facets of QOL that also need to be considered. The worst physical symptom on admission is the item of QOL with the lowest score, which need more care and attention by palliative care workers. A cross-culturally validated QOL instrument cannot just help ensure an accurate evaluation of profile, determinants, and changes of QOL, but is also a valuable asset for future comparison and evaluation of palliative care services and interventions across the world.

Epidermal growth factor signaling via Ras controls the Smad transcriptional co-repressor TGIF.

Smad transcription factors mediate the actions of transforming growth factor-beta (TGF-beta) cytokines during development and tissue homeostasis. TGF-beta receptor-activated Smad2 regulates gene expression by associating with transcriptional co-activators or co-repressors. The Smad co-repressor TGIF competes with the co-activator p300 for Smad2 association, such that TGIF abundance helps determine the outcome of a TGF-beta response. Small alterations in the physiological levels of TGIF can have profound effects on human development, as shown by the devastating brain and craniofacial developmental defects in heterozygotes carrying a hypomorphic TGIF mutant allele. Here we show that TGIF levels modulate sensitivity to TGF-beta-mediated growth inhibition, that TGIF is a short-lived protein and that epidermal growth factor (EGF) signaling via the Ras-Mek pathway causes the phosphorylation of TGIF at two Erk MAP kinase sites, leading to TGIF stabilization and favoring the formation of Smad2-TGIF co-repressor complexes in response to TGF-beta. These results identify the first mechanism for regulating TGIF levels and suggest a potential link for Smad and Ras pathway convergence at the transcriptional level.

Disaster psychosocial services in Hong Kong--make the unseen injury seen.

While intervention services for the physical consequences of disaster and trauma have been in evidence for decades, only within recent years has serious attention been paid to the psychological consequences of disaster. This paper describes the development of a program of psychosocial services designed to respond to the needs of the people of Hong Kong in the wake of a disaster.

Multiple modes of repression by the Smad transcriptional corepressor TGIF.

TGIF is a DNA-binding homeodomain protein that has been demonstrated to play a role in transforming growth factor beta-regulated transcription and implicated in the control of retinoid-responsive transcription. We investigated the intrinsic transcriptional activity of TGIF fused to a heterologous DNA-binding domain. Our results demonstrate that TGIF is a transcriptional repressor able to repress transcription from several different promoters. Repression by TGIF is insensitive to the distance at which it is bound from the promoter. Moreover, the wild type TGIF effectively represses transcription when bound to its cognate DNA-binding site via its homeodomain. Deletion analysis revealed the presence of at least two separable repression domains within TGIF. Repression by one of these is dependent on the activity of histone deacetylases (HDACs), whereas the other appears not to require HDAC activity. Finally, we demonstrate that TGIF interacts with HDACs via its carboxyl-terminal repression domain. Together, these results suggest that TGIF is a multifunctional transcriptional repressor, which acts in part by recruiting HDAC activity.

Ubiquitin-dependent degradation of TGF-beta-activated smad2.

SMAD proteins are phosphorylated by transforming growth factor-beta (TGF-beta) receptors and translocate to the nucleus, where they control transcription. Here we investigate the fate of activated Smad2. We show that receptor-mediated activation leads to multi-ubiquitination and subsequent degradation of Smad2 by the proteasome. Ubiquitination of Smad2 is a consequence of its accumulation in the nucleus. If degradation is averted, the phosphorylated Smad2 remains in the nucleus in an active state. By targeting Smad2 for destruction, TGF-beta ensures the irreversible termination of its own signalling function.

TbetaR-I(6A) is a candidate tumor susceptibility allele.

We have previously described a type I transforming growth factor (TGF)-beta receptor (TbetaR-I) polymorphic allele, TbetaR-I(6A), that has a deletion of three alanines from a nine-alanine stretch. We observed a higher than expected number of TbetaR-I(6A) homozygotes among tumor and nontumor DNA from patients with a diagnosis of cancer. To test the hypothesis that TbetaR-I(6A) homozygosity is associated with cancer, we performed a case-control study in patients with a diagnosis of cancer and matched healthy individuals with no history of cancer and who were identical in their gender and their geographical and ethnic background to determine the relative germ-line frequencies of this allele. We found nine TbetaR-I(6A) homozygotes among 851 patients with cancer. In comparison, there were no TbetaR-I(6A) homozygotes among 735 healthy volunteers (P < 0.01). We also observed an excess of TbetaR-I(6A) heterozygotes in cancer cases compared to controls (14.6% versus 10.6%; P = 0.02, Fisher's exact test). A subset analysis revealed that 4 of 112 patients with colorectal cancer were TbetaR-I(6A) homozygotes (P < 0.01). Using mink lung epithelial cell lines devoid of TbetaR-I, we established stably transfected TbetaR-I and TbetaR-I(6A) cell lines. We found that, compared to TbetaR-I, TbetaR-I(6A) was impaired as a mediator of TGF-beta antiproliferative signals. We conclude that TbetaR-I(6A) acts as a tumor susceptibility allele that may contribute to the development of cancer, especially colon cancer, by means of reduced TGF-beta-mediated growth inhibition.

A Smad transcriptional corepressor.

Following TGFbeta receptor-mediated phosphorylation and association with Smad4, Smad2 moves into the nucleus, binds to target promoters in association with DNA-binding cofactors, and recruits coactivators such as p300/CBP to activate transcription. We identified the homeodomain protein TGIF as a Smad2-binding protein and a repressor of transcription. A TGFbeta-activated Smad complex can recruit TGIF and histone deacetylases (HDACs) to a Smad target promoter, repressing transcription. Thus, upon entering the nucleus, a Smad2-Smad4 complex may interact with coactivators, forming a transcriptional activation complex, or with TGIF and HDACs, forming a transcriptional repressor complex. Formation of one of these two mutually exclusive complexes is determined by the relative levels of Smad corepressors and coactivators within the cell.

Prospective study of symptom control in 133 cases of palliative care inpatients in Shatin Hospital.

We report a prospective study assessing the prevalence and severity of physical and nonphysical symptoms, and the benefits from treatment and intervention, in advanced cancer patients presenting to a local palliative care unit in Hong Kong. Patients were assessed by a modified version of the support team assessment schedule (STAS). The study highlighted some symptoms which needed better control, and also reinforced the team morale by demonstrating aspects where there was definite improvement. In general, the STAS was found to be practicable and acceptable by our patients and staff. The most important benefit gained from the study was the successful dissemination of the concept of audit and quality assurance throughout the unit, which is essential for continuous improvement in the future.

Determinants of specificity in TGF-beta signal transduction.

Signal transduction by the TGF-beta family involves sets of receptor serine/threonine kinases, Smad proteins that act as receptor substrates, and Smad-associated transcription factors that target specific genes. We have identified discrete structural elements that dictate the selective interactions between receptors and Smads and between Smads and transcription factors in the TGF-beta and BMP pathways. A cluster of four residues in the L45 loop of the type I receptor kinase domain, and a matching set of two residues in the L3 loop of the Smad carboxy-terminal domain establish the specificity of receptor-Smad interactions. A cluster of residues in the highly exposed alpha-helix 2 of the Smad carboxy-terminal domain specify the interaction with the DNA-binding factor Fast1 and, as a result, the gene responses mediated by the pathway. By establishing specific interactions, these determinants keep the TGF-beta and BMP pathways segregated from each other.

The L3 loop: a structural motif determining specific interactions between SMAD proteins and TGF-beta receptors.

Signal transduction specificity in the transforming growth factor-beta (TGF-beta) system is determined by ligand activation of a receptor complex which then recruits and phosphorylates a subset of SMAD proteins including Smads 1 and 2. These then associate with Smad4 and move into the nucleus where they regulate transcription. We have identified a discrete surface structure in Smads 1 and 2 that mediates and specifies their receptor interactions. This structure is the L3 loop, a 17 amino acid region that protrudes from the core of the conserved SMAD C-terminal domain. The L3 loop sequence is invariant among TGF-beta- and bone morphogenetic protein (BMP)-activated SMADS, but differs at two positions between these two groups. Swapping these two amino acids in Smads 1 and 2 induces a gain or loss, respectively, in their ability to associate with the TGF-beta receptor complex and causes a switch in the phosphorylation of Smads 1 and 2 by the BMP and TGF-beta receptors, respectively. A full switch in phosphorylation and activation of Smads 1 and 2 is obtained by swapping both these two amino acids and four amino acids near the C-terminal receptor phosphorylation sites. These studies identify the L3 loop as a determinant of specific SMAD-receptor interactions, and indicate that the L3 loop, together with the C-terminal tail, specifies SMAD activation.

Mutations increasing autoinhibition inactivate tumour suppressors Smad2 and Smad4.

Smad2 and Smad4 are related tumour-suppressor proteins, which, when stimulated by the growth factor TGF-beta, form a complex to inhibit growth. The effector function of Smad2 and Smad4 is located in the conserved carboxy-terminal domain (C domain) of these proteins and is inhibited by the presence of their amino-terminal domains (N domain). This inhibitory function of the N domain is shown here to involve an interaction with the C domain that prevents the association of Smad2 with Smad4. This inhibitory function is increased in tumour-derived forms of Smad2 and 4 that carry a missense mutation in a conserved N domain arginine residue. The mutant N domains have an increased affinity for their respective C domains, inhibit the Smad2-Smad4 interaction, and prevent TGF beta-induced Smad2-Smad4 association and signalling. Whereas mutations in the C domain disrupt the effector function of the Smad proteins, N-domain arginine mutations inhibit SMAD signalling through a gain of autoinhibitory function. Gain of autoinhibitory function is a new mechanism for inactivating tumour suppressors.

A structural basis for mutational inactivation of the tumour suppressor Smad4.

The Smad4/DPC4 tumour suppressor is inactivated in nearly half of pancreatic carcinomas and to a lesser extent in a variety of other cancers. Smad4/DPC4, and the related tumour suppressor Smad2, belong to the SMAD family of proteins that mediate signalling by the TGF-beta/activin/BMP-2/4 cytokine superfamily from receptor Ser/Thr protein kinases at the cell surface to the nucleus. SMAD proteins, which are phosphorylated by the activated receptor, propagate the signal, in part, through homo- and hetero-oligomeric interactions. Smad4/DPC4 plays a central role as it is the shared hetero-oligomerization partner of the other SMADs. The conserved carboxy-terminal domains of SMADs are sufficient for inducing most of the ligand-specific effects, and are the primary targets of tumorigenic inactivation. We now describe the crystal structure of the C-terminal domain (CTD) of the Smad4/DPC4 tumour suppressor, determined at 2.5 A resolution. The structure reveals that the Smad4/DPC4 CTD forms a crystallographic trimer through a conserved protein-protein interface, to which the majority of the tumour-derived missense mutations map. These mutations disrupt homo-oligomerization in vitro and in vivo, indicating that the trimeric assembly of the Smad4/DPC4 CTD is critical for signalling and is disrupted by tumorigenic mutations.

A conserved glutamate is responsible for ion selectivity and pH dependence of the mammalian anion exchangers AE1 and AE2.

The erythrocyte anion exchanger AE1 (band 3) serves as an important model for the study of the mechanism of ion transport. Chemical modification of human erythrocyte AE1 has previously suggested that glutamic acid residue 681 lies within the transport pathway and can cross the permeability barrier. This glutamate is conserved in all anion exchangers sequenced to date. We examined the effect on divalent (sulfate) and monovalent (chloride and bicarbonate) anion transport of mutating the corresponding glutamates in mouse AE1 and the closely related anion exchanger, AE2. Substitution of this conserved glutamate with uncharged or basic amino acids had a negligible effect on the maximal rate of sulfate-sulfate exchange in AE-reconstituted proteoliposomes, but largely abolished the steep pH dependence of sulfate transport observed in wild-type AE1 and AE2. In contrast, exchange of monovalent anions was undetectable in cells expressing these mutants. Replacement of the conserved glutamate with aspartate abolished both monovalent and divalent anion transport. These data suggest that the conserved glutamate residue plays a dual role in determining anion selectivity and in proton coupling to sulfate transport. A model explaining the role of the conserved glutamate in promoting ion selectivity and pH regulation is discussed.

Sulfate transport mediated by the mammalian anion exchangers in reconstituted proteoliposomes.

The kinetic properties of sulfate transport mediated by the anion exchangers AE1 and AE2 have been examined. Microsomes isolated from HEK cells transiently overexpressing either protein were reconstituted in unilamellar, 200-600-nm diameter proteoliposomes. Transport mediated by the exchangers was monitored by loading the reconstituted proteoliposomes with the slowly transportable anion SO4(2-) using [35S]SO4(2-) as a tracer and performing [35S]SO4(2-)/SO4(2-) exchange. The following data suggest that AE1 and AE2 have been functionally reconstituted: (i) the rate of SO4(2-) transport in AE1 and AE2 containing proteoliposomes was 10-20 times higher than in proteoliposomes derived from control microsomes; (ii) the transport of SO4(2-) was strongly dependent on the presence of a trans anion; and (iii) the anion exchanger inhibitors, 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 4,4'-dinitrostilbene-2,2'-di-sulfonate (DIDS) totally abolished SO4(2-) transport. furthermore, DIDS inhibits SO4(2-) transport only when occluded inside the vesicles, indicating a uniform, asymmetrical, inside-out orientation of the reconstituted exchangers. The Ki values of the stilbene disulfonate compound DNDS were 2.5 and 4 microM for AE1 and AE2, respectively, suggesting that the two exchangers possess similar high affinity sites for stilbene compounds. Both AE1 and AE2 showed the same steep pH dependence of sulfate transport, which was maximal at pH 5.5 and reduced to less than 10% (of the value at pH 5.5) at pH 8.5, suggesting that an acidic residue shared by AE1 and AE2 participates in the pH regulation of sulfate transport.