James Howard Pringle

Vitamin D receptor gene polymorphisms, particularly the novel A-1012g promoter polymorphism, are associated with vitamin D3 responsiveness and non-familial susceptibility in psoriasis

Psoriasis is a genetically determined disease characterized by hyperproliferation and disordered maturation of the epidermis. Th1 lymphocytes are implicated in its pathogenesis. The vitamin D receptor (VDR) is a candidate modifying gene, having immunosuppressive effects and being involved in anti-proliferative and pro-differentiation pathways in keratinocytes. There is suggestive evidence that the A allele of the A-1012G polymorphism is associated with down-regulation of the Th1 response, via GATA-3. The F and T alleles of Fok1 and Taq1 have been associated with increased VDR activity. The present study aimed to test the hypothesis that the A allele of A-1012G is protective for occurrence and severity of psoriasis and enhances therapeutic response to vitamin D analogues and that these effects would be additive to those of Fok1 and Taq1. The study group comprised 206 psoriasis patients who had received topical calcipotriol treatment and 80 controls. There was no significant linkage disequilibrium between any pair of the three polymorphic sites (P=0.3–0.8). The A, F and T alleles were positively associated with calcipotriol response: AA genotype (compared to AG/GG), odds ratio (OR)=2.18 (P=0.04); TT, OR=1.97 (P=0.03); AAFF genotype combination, OR=4.11 (P=0.03); AATT, OR=5.64 (P=0.005); and FFTT, OR=3.22 (P=0.01). Comparing patients without, to patients with, a family history of psoriasis, the A allele was under represented (P=0.01) and the AAFF genotype combination even more so (compared to residual genotypes) (OR=0.24; P=0.005). AAFF was also under-represented in patients without a family history compared to controls (OR=0.31; P=0.04). There were no associations of family history with Fok1 and Taq1. There were no associations of severity of psoriasis with any polymorphism. In conclusion, the A-1012G, Fok1 and Taq1 VDR polymorphisms were associated with response to calcipotriol. A-1012G and Fok1 were associated with susceptibility to non-familial psoriasis.

Characterisation of Kir2.0 proteins in the rat cerebellum and hippocampus by polyclonal antibodies.

Abstractu2002Rabbit polyclonal antibodies were raised to rat Kir2.0 (Kir2.1, Kir2.2 and Kir2.3) inwardly rectifying potassium ion channel proteins. The antibody specificities were confirmed by immunoprecipitation of [35S]-methionine-labelled in vitro translated channel proteins and western blotting. Immunohistochemistry revealed a different patterns of expression of Kir2.0 subfamily proteins in the rat hind-brain (cerebellum and medulla) and fore-brain (hippocampus). Notably, only Kir2.2 protein was detected in the cerebellum and medulla, Kir2.1, Kir2.2 and Kir2.3 proteins were expressed in the hippocampus and immunostaining was not limited to neuronal cell types. Anti-Kir2.1 (fore-brain only) and anti-Kir2.2 (fore- and hind-brain) antibodies showed positive staining in macroglia, endothelia, ependyma and vascular smooth muscle cells. In contrast, anti-Kir2.3 (fore-brain only) immunostaining was limited to neurons, macroglia and vascular smooth muscle. These results indicate that specific regions within the rat fore- and hind-brain have differential distributions of inwardly rectifying potassium ion channel proteins.

Ectopic Expression of P-Cadherin Correlates with Promoter Hypomethylation Early in Colorectal Carcinogenesis and Enhanced Intestinal Crypt Fission In vivo

P-cadherin is normally expressed in the basal layer of squamous epithelia and absent from the healthy intestine and colon. We have previously shown it to be expressed in all inflamed, hyperplastic, and dysplastic intestinal and colonic mucosa. This study aimed to better understand the mechanisms controlling the expression of P-cadherin and the biological effects of its ectopic presence in the intestine and colon. We investigated the CpG methylation status of the P-cadherin (CDH3) promoter and P-cadherin mRNA and protein expression in cases of familial and sporadic colorectal cancer (CRC). The CDH3 promoter was hypomethylated in colonic aberrant crypt foci, in CRC, and, occasionally, in the normal epithelium adjacent to cancer, demonstrating a potential field effect of cancerization. The hypomethylation was also associated with induction of P-cadherin expression in the neoplastic colon (P < 0.0001). We then created transgenic mice that overexpressed P-cadherin specifically in the intestinal and colonic epithelium under the liver fatty acid binding protein promoter. Forced ectopic expression of P-cadherin accompanied by indomethacin-induced inflammation resulted in a 3-fold higher crypt fission rate within the small and large intestines in the homozygous mice compared with the wild-type animals (P < 0.02). We conclude that epigenetic demethylation of the P-cadherin promoter in the human intestine permits its ectopic expression very early in the colorectal adenoma-carcinoma sequence and persists during invasive cancer. Induced P-cadherin expression, especially in mucosal damage, leads to an increased rate of crypt fission, a common feature of clonal expansion in gastrointestinal dysplasia.

Uterine adenomyosis is associated with ultrastructural features of altered contractility in the inner myometrium.

OBJECTIVEnTo study the ultrastructure of the inner and outer myometrium, in the presence and absence of uterine adenomyosis.nnnDESIGNnCase control blinded comparison.nnnSETTINGnUniversity departments.nnnPATIENT(S)nFour premenopausal women with and six without uterine adenomyosis as the sole pathology.nnnINTERVENTION(S)nMultiple samples were studied using transmission electron microscopy.nnnMAIN OUTCOME MEASURE(S)nUltrastructure feature of the myometrium.nnnRESULT(S)nIn uteri with adenomyosis, the myocytes exhibited cellular hypertrophy. The cytoplasmic myofilaments were less abundant. Abundant intermediate filaments formed cytoplasmic aggregates. The nuclei had a smooth outline with a clear ground substance, prominent nucleoli and peripherally arranged nuclear chromatin. There was occasional infolding of the nuclear envelope with entrapment of cytoplasmic organelles. The sarcolemmal bands were significantly longer and there were fewer caveolae. The perinuclear cell organelles were more distinct. The rough endoplasmic reticulum and Golgi apparatus were more prominent, denoting active protein synthesis, consistent with the observed cellular hypertrophy. All features were more prominent at the junctional zone.nnnCONCLUSION(S)nSmooth muscle cells from uteri with adenomyosis are ultrastructurally different from smooth muscle cells of normal uteri. These distinct features suggest a possible effect on myometrial contractility, together with hypertrophy.

Phenotypic characterisation of the inner and outer myometrium in normal and adenomyotic uteri.

Background and Aim: To study the characteristics of the inner (IM) and outer (OM) myometrium in the presence and absence of uterine adenomyosis. Methods: Case control blinded comparison carried out in a university department. Morphometric features of the myometrium were studied in uteri from pre- and postmenopausal women with and without uterine adenomyosis as the sole pathology. Uteri were also divided according to the phase of the cycle and examined using immunohistochemistry and image analysis. Results: Cell density and total nuclear area were statistically significantly greater in the IM compared to OM, in pre- and postmenopausal women, in both the adenomyosis and control uteri. The difference in nuclear size was statistically significant only in the premenopausal group. The change from the IM to the OM in cell density and total nuclear area was gradual with no distinct zonation. Examined features did not vary with cycle phase. Both the IM and OM in adenomyosis exhibited lower cell density and larger nuclei compared to controls. In adenomyosis, immunostaining for α-smooth muscle actin, desmin and Ki-67 was consistent with myometrial hyperplasia and hypertrophy. Conclusions: There are clear differences between the IM and the OM but the transition is gradual, with no distinct zonation. Adenomyosis is characterised by reduced cell density, and increased nuclear size and features of hyperplasia and hypertrophy that are not confined to the IM.

The unfavorable effect of the A allele of the vitamin D receptor promoter polymorphism A-1012G has different mechanisms related to susceptibility and outcome of malignant melanoma.

The A allele of the A-1012G (rs4516035) vitamin D receptor (VDR) promoter polymorphism is associated with increased susceptibility and worsened outcome in malignant melanoma (MM). The A allele contains a GATA-3 binding site. There is a second polymorphism in the same promoter region, G-1520C (rs7139166), and there is potential for another GATA binding site in the G allele. Here, we tested the hypothesis that the G-1520A-1012 haplotype might be a greater risk factor for MM than A-1012 alone. The A allele of A-1012G was preferentially linked to G of G-1520C and was more frequent in MM patients (P=0.011) but G of G-1520C was not (P=0.756). The CA haplotype was a very significant risk factor for MM (P=0.0001) while the CG haplotype was protective (P=0.014, combined model P=0.00002). There was no effect of GA haplotype (P=0.931), suggesting that that the difference in frequencies of the A allele between patients and controls was accounted for by the differences in frequencies of the CA haplotype. The A allele of A-1012G was more frequent in patients with metastasis (P=0.054) than MM patients without metastasis, as was the G allele of G-1520C (P=0.028). The GA haplotype was more frequent in patients with metastasis (P=0.015), while frequencies of CA were similar. We suggest that the different roles of the A allele of A-1012G in susceptibility and metastasis risk may be a function of the availability of transcription factors in the differing cellular backgrounds related to susceptibility and progression of MM.

Enhanced invasion of stromal cells from adenomyosis in a three-dimensional coculture model is augmented by the presence of myocytes from affected uteri

OBJECTIVEnTo compare endometrial stromal cell invasion from women with and without adenomyosis and the effect of myometrial cells using a three-dimensional coculture.nnnDESIGNnCase-controlled blinded comparison.nnnSETTINGnUniversity department.nnnPATIENT(S)nPremenopausal women with and without uterine adenomyosis.nnnINTERVENTION(S)nHuman endometrial stromal and myometrial cells were grown in a three-dimensional coculture with crossover between cells from uteri with and without adenomyosis. Surface-enhanced laser desorption/ionization-time of flight-mass spectrometry proteomic analysis was performed on culture supernatants.nnnMAIN OUTCOME MEASURE(S)nDepth of stromal cell invasion into collagen matrix and protein expression profiles.nnnRESULT(S)nThe depth of invasion for adenomyosis stromal cells (AS) was statistically significantly higher than controls (CS) whether grown on plain collagen, control muscle (CM), or adenomyosis muscle (AM). Coculture with AM enhanced invasion of both CS and AS. Enhanced invasion by AS was more marked in cocultures with AM than CM. Proteomic analysis identified differences that may account for the invasiveness and also many similarities between secretory products related to the disease status.nnnCONCLUSION(S)nEnhanced stromal invasion in adenomyosis is influenced by the myometrium in the in vitro coculture model. This suggests that adenomyosis may be a disease of both the endometrial stroma and myometrium.

Circulating plasma microRNAs as a screening method for detection of colorectal adenomas.

BACKGROUNDnMicroRNAs (miRNAs) are small non-coding RNA molecules. Reduced or increased levels of specific miRNAs are observed in colon and other cancers, supporting their role in carcinogenesis. Detection of colorectal polyps is the cornerstone of the Bowel Cancer Screening Programme in the UK. However, uptake of screening nationally remains under 60%. We aimed to see whether circulating plasma miRNAs can be used to screen for patients with colorectal polyps, adenomas, or both.nnnMETHODSnBlood samples were taken from patients from the Bowel Cancer Screening Programme (asymptomatic but faecal occult blood testing [FOBt] positive). Plasma RNA was extracted, target miRNAs (19a, 98, 146b, 186, 191, 222*, 331-5p, 452, 625, 664, 1247) were identified on pooled case miRNA assay cards, and miRNA fraction was quantified by quantitative RT-PCR assay. Results were compared with endoscopy reports and with histology of any polyps identified and removed. Analysis was done with Excel (2011) and SPSS (version 20) software.nnnFINDINGSn210 patients were included (117 with polyps, 12 with cancer, 81 healthy controls [FOBt positive]). The miRNA panel showed significant differences in expression (on t testing) for patients compared with controls for those with polyps, cancer, or both (miR-19a, p=0·0184; miR-98, p=0·0206; miR-146b, p=0·0029; miR-186, p=0·0006; miR-62,5 p=0·0008), polyps (miR-19a, p=0·0233; miR-98, p=0·0224; miR-146b, p=0·003; miR-186, p=0·0004; miR-625, p=0·001), adenomas (miR-19a, p=0·0339; miR-98, p=0·0266; miR-146b, p=0·0045; miR-186, p=0·0008; miR-625, p=0·0049), multiple adenomas (both sides of colon; miR-146b, p=0·0194; miR-186, p=0·0226; miR-625, p=0·0013), and right-sided adenomas (miR-98, p=0·031; miR-146b, p=0·0076; miR-186, p=0·0041; miR-331-5p, p=0·0142; miR-625, p=0·0049). Receiver operating characteristic analysis showed sensitivity of 60% or more, and specificity of 86% or more for men with polyps, men with adenomas, all patients with haemorrhoids or diverticulosis and polyps, and all patients with haemorrhoids or diverticulosis and adenomas.nnnINTERPRETATIONnThe target miRNAs that we identified showed significant differences in expression levels for patients with polyps and patients with adenomas from controls. Use of this panel has potential as a screening test.nnnFUNDINGnBowel Disease Research Foundation.

Higher Serum 25-Hydroxy Vitamin D3 Levels at Presentation Are Associated With Improved Survival From Melanoma, But There Is No Evidence That Later Prevailing Levels Are Protective

TO THE EDITOR: We read with interest the article by NewtonBishop et al, which provides further clinical evidence that vitamin D has an effect on cutaneous melanoma (MM) outcome, as has been suggested by previous studies on vitamin D receptor (VDR) polymorphisms. There is a strong body of epidemiologic evidence that vitamin D status has a protective effect against major systemic cancers. The authors report two studies: the first, a retrospective study, was described as a pilot study for the second prospective study, though surprisingly, there was a long overlap period between the two. The retrospective study involved patients with at least a 3-year, relapse-free period following initial diagnosis of the MM, and serum for 25hydroxyvitamin D3 [25(OH)D3] was taken on patients’ entry onto the study; the median time after diagnosis was 6.5 to 7.5 years. There was no statistically significant difference in serum 25(OH)D3 between patients who did or did not experience relapse (P .3). In the prospective study, serum 25(OH)D3 was estimated at a time close to presentation of the primary MM, and the serum was found to have a protective effect on Breslow thickness and an independent beneficial effect on relapse and survival. The serum 25(OH)D3 at presentation is likely to be a reflection of levels before development of the MM and during early development of the MM; therefore, vitamin D appears to inhibit local invasion and micrometastases during early tumor development. This certainly underlines the need to maintain vitamin D levels in the community. The authors go on to state that “. . . patients with melanoma who are avoiding sun exposure should take vitamin D3 supplements sufficient to ensure normal levels.” There are two implications of this: first, the authors believe that the prevailing vitamin D status after excision of the primary MM has an effect on survival in addition to that of the vitamin D status at presentation; and second, vitamin D is still protective during that phase. We would support the first contention, but we believe the second is unjustified. It seems likely that the development of clinical metastases is a function of vitamin D status during early development of the tumor, during local invasion and development of micrometastases, and after presentation, during the phase of growth of micrometastases into clinical metastases. Later vitamin D status was not investigated in the prospective study, but there were limited, relevant data in the retrospective study. In that study, there was no statistically significant difference in serum 25(OH)D3 between patients who experienced relapse and those who did not, and the levels were apparently lower than those at presentation of the MM in the prospective study. This would suggest that, at the time (median 6.6 years after diagnosis), 25(OH)D3 levels were neither protective against nor a risk factor for relapse. However, the group of patients who experienced relapse was atypical in that relapses occurred late for a large majority of patients. The study entry criterion was that a patient had experienced no relapse within 3 years, and the mean time to relapse was 6.6 years, whereas the majority of relapses in an unselected series of MM occurs within 5 years. Thus no information is available for serum 25(OH)D3 levels up to when the large majority of relapses occurs. The fact that later levels in the retrospective study were lower than the levels at presentation of the prospective study might provide some evidence that higher 25(OH)D3 after excision predisposes to relapse. A greater proportion of patients with higher serum levels could have experienced relapse before the entry point of the retrospective study, leaving a population of patients with melanomas with lower levels of 25(OH)D3 still eligible for trial entry. An alternative explanation for lower levels in the retrospective study is that patients avoid sun exposure after diagnosis. Without further evidence, it is not possible to know which explanation is true or whether both are true. It is rather surprising that the authors made no estimate of levels of sun exposure in either study, given that sun exposure is accepted as a more important source of vitamin D than are dietary supplements, which they did investigate extensively. There are strong theoretical reasons for believing that vitamin D status after MM excision may not be protective but may predispose to further progression through immune suppression. The immunosuppressive effects of vitamin D include inhibition of dendritic cells, as mentioned by the authors, and a consequent downregulation of the T-helper cell 1 response and a direct inhibitory effect on T lymphocytes. There have also been reports of downregulation of natural killer cells. The net effect of vitamin D in cancer may be a balance between beneficial anticancer effects on the tumor cells and deleterious effects via the immune system. The authors imply that higher vitamin D levels may favor the immune-inhibitory effects. We are not aware of this, but we would suggest that a loss of expression of the VDR in the tumor cells would critically impair the anticancerous effect of vitamin D, leaving immune suppression unopposed. We have immunohistochemical evidence (unpublished) that VDR protein expression is reduced in MM, particularly in thick tumors, and more so in metastases. Similarly, in more aggressive colon cancer, decreased VDR expression has been reported as well as very low expression in metastases. Furthermore, there is evidence that response to vitamin D is compromised when VDR is poorly expressed in MM cell lines. In conclusion, many previous studies support the hypothesis that vitamin D has a protective effect on the development of cancer. Indeed, the article by Newton-Bishop et al adds weight to this and would suggest a beneficial effect in the early stages of development of cancer. However, we believe it is important not to extrapolate these results to a beneficial effect of the prevailing vitamin D status later (ie, following excision of the primary). There is no evidence from Newton-Bishop et al that, in this phase of metastatic growth, vitamin D is advantageous, and there is reason to suspect that it may be disadvantageous in the presence of downregulated tumor cell VDR. JOURNAL OF CLINICAL ONCOLOGY C O R R E S P O N D E N C E VOLUME 28 NUMBER 27 SEPTEMBER 2

Resistance to HSP90 inhibition involving loss of MCL1 addiction

Inhibition of the chaperone heat-shock protein 90 (HSP90) induces apoptosis, and it is a promising anti-cancer strategy. The mechanisms underpinning apoptosis activation following HSP90 inhibition and how they are modified during acquired drug resistance are unknown. We show for the first time that, to induce apoptosis, HSP90 inhibition requires the cooperation of multi BH3-only proteins (BID, BIK, PUMA) and the reciprocal suppression of the pro-survival BCL-2 family member MCL1, which occurs via inhibition of STAT5A. A subset of tumour cell lines exhibit dependence on MCL1 expression for survival and this dependence is also associated with tumour response to HSP90 inhibition. In the acquired resistance setting, MCL1 suppression in response to HSP90 inhibitors is maintained; however, a switch in MCL1 dependence occurs. This can be exploited by the BH3 peptidomimetic ABT737, through non-BCL-2-dependent synthetic lethality.