Targeting LGR5 in Colorectal Cancer: therapeutic gold or too plastic?

Leucine-rich repeat-containing G-protein coupled receptor (LGR5 or GPR49) potentiates canonical Wnt/ß-catenin signalling and is a marker of normal stem cells in several tissues, including the intestine. Consistent with stem cell potential, single isolated LGR5+ cells from the gut generate self-organising crypt/villus structures in vitro termed organoids or 'mini-guts', which accurately model the parent tissue. The well characterised deregulation of Wnt/ß-catenin signalling that occurs during the adenoma-carcinoma sequence in colorectal cancer (CRC) renders LGR5 an interesting therapeutic target. Furthermore, recent studies demonstrating that CRC tumours contain LGR5+ subsets and retain a degree of normal tissue architecture has heightened translational interest. Such reports fuel hope that specific subpopulations or molecules within a tumour may be therapeutically targeted to prevent relapse and induce long-term remissions. Despite these observations, many studies within this field have produced conflicting and confusing results with no clear consensus on the therapeutic value of LGR5. This review will recap the various oncogenic and tumour suppressive roles that have been described for the LGR5 molecule in CRC. It will further highlight recent studies indicating the plasticity or redundancy of LGR5+ cells in intestinal cancer progression and assess the overall merit of therapeutically targeting LGR5 in CRC.

Optimized delivery of siRNA into 3D tumor spheroid cultures in situ.

3D tissue culture provides a physiologically relevant and genetically tractable system for studying normal and malignant human tissues. Despite this, gene-silencing studies using siRNA has proved difficult. In this study, we have identified a cause for why traditional siRNA transfection techniques are ineffective in eliciting gene silencing in situ within 3D cultures and proposed a simple method for significantly enhancing siRNA entry into spheroids/organoids. In 2D cell culture, the efficiency of gene silencing is significantly reduced when siRNA complexes are prepared in the presence of serum. Surprisingly, in both 3D tumour spheroids and primary murine organoids, the presence of serum during siRNA preparation rapidly promotes entry and internalization of Cy3-labelled siRNA in under 2 hours. Conversely, siRNA prepared in traditional low-serum transfection media fails to gain matrigel or spheroid/organoid entry. Direct measurement of CTNNB1 mRNA (encoding ß-catenin) from transfected tumour spheroids confirmed a transient but significant knockdown of ß-catenin when siRNA:liposome complexes were formed with serum, but not when prepared in the presence of reduced-serum media (Opti-MEM). Our studies suggest a simple modification to standard lipid-based transfection protocols facilitates rapid siRNA entry and transient gene repression, providing a platform for researchers to improve siRNA efficiency in established 3D cultures.

LGR5 expression is regulated by EGF in early colorectal adenomas and governs EGFR inhibitor sensitivity.

BACKGROUND: LGR5 serves as a co-receptor for Wnt/ß-catenin signalling and marks normal intestinal stem cells; however, its role in colorectal cancer (CRC) remains controversial. LGR5+ cells are known to exist outside the stem cell niche during CRC progression, and the requirement for epidermal growth factor (EGF) signalling within early adenomas remains to be fully elucidated. METHODS: Epidermal growth factor and gefitinib treatments were performed in EGF-responsive LGR5+ early adenoma RG/C2 cells. 2D growth assays were measured using an IncuCyte. LGR5 or MEK1/2 silencing studies were executed using siRNA and LGR5 expression was assessed by qRT-PCR and immunoblotting. Ki67 level and cell cycle status were analysed by flow cytometry. RESULTS: Epidermal growth factor suppresses expression of LGR5 at both the transcript and protein level in colorectal adenoma and carcinoma cells. Suppression of LGR5 reduces the survival of EGF-treated adenoma cells by increasing detached cell yield but also inducing a proliferative state, as evidenced by elevated Ki67 level and enhanced cell cycle progression. Repression of LGR5 further increases the sensitivity of adenoma cells to EGFR inhibition. CONCLUSIONS: LGR5 has an important role in the EGF-mediated survival and proliferation of early adenoma cells and could have clinical utility in predicting response of CRC patients to EGFR therapy.

Characterization of cognitive impairments and neurotransmitter changes in a novel transgenic mouse lacking Slc10a4.

An orphan member of the solute carrier (SLC) family SLC10, SLC10A4 has been found to be enriched in midbrain and brainstem neurons and has been found to co-localize with and to affect dopamine (DA) homeostasis. We generated an SLC10A4 knockout mouse (Slc10a4(Δ/Δ)) using Cre-targeted recombination, and characterized behavioral measures of motor and cognitive function as well as DA and acetylcholine (ACh) levels in midbrain and brainstem. In agreement with previous studies, Slc10a4 mRNA was preferentially expressed in neurons in the brains of wild-type (Slc10a4(+/+)) mice and was enriched in dopaminergic and cholinergic regions. Slc10a4(Δ/Δ) mice had no impairment in motor function or novelty-induced exploratory behaviors but performed significantly worse in measures of spatial memory and cognitive flexibility. Slc10a4(Δ/Δ) mice also did not differ from Slc10a4(+/+) in measures of anxiety. High-performance liquid chromatography (HPLC) measures on tissue punches taken from the dorsal and ventral striatum reveal a decrease in DA content and a corresponding increase in the metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), indicating an increase in DA turnover. Punches taken from the brainstem revealed a decrease in ACh as compared with Slc10a4(+/+) littermates. Together, these data indicate that loss of SLC10A4 protein results in neurotransmitter imbalance and cognitive impairment.

Nutrient stress alters the glycosylation status of LGR5 resulting in reduced protein stability and membrane localisation in colorectal tumour cells: implications for targeting cancer stem cells.

BACKGROUND: LGR5 is an important marker of intestinal stem cells and performs its vital functions at the cell membrane. Despite the importance of LGR5 to both normal and cancer stem cell biology, it is not known how microenvironmental stress affects the expression and subcellular distribution of the protein. METHODS: Nutrient stress was induced through glucose starvation. Glycosylation status was assessed using endoglycosidase or tunicamycin treatment. Flow cytometry and confocal microscopy were used to assess subcellular distribution of LGR5. RESULTS: Glucose deprivation altered the glycosylation status of LGR5 resulting in reduced protein stability and cell surface expression. Furthermore, inhibiting LGR5 glycosylation resulted in depleted surface expression and reduced localisation in the cis-Golgi network. CONCLUSIONS: Nutrient stress within a tumour microenvironment has the capacity to alter LGR5 protein stability and membrane localisation through modulation of LGR5 glycosylation status. As LGR5 surface localisation is required for enhanced Wnt signalling, this is the first report to show a mechanism by which the microenvironment could affect LGR5 function.

γ-Catenin is overexpressed in acute myeloid leukemia and promotes the stabilization and nuclear localization of ß-catenin.

Canonical Wnt signaling regulates the transcription of T-cell factor (TCF)-responsive genes through the stabilization and nuclear translocation of the transcriptional co-activator, ß-catenin. Overexpression of ß-catenin features prominently in acute myeloid leukemia (AML) and has previously been associated with poor clinical outcome. Overexpression of γ-catenin mRNA (a close homologue of ß-catenin) has also been reported in AML and has been linked to the pathogenesis of this disease, however, the relative roles of these catenins in leukemia remains unclear. Here we report that overexpression and aberrant nuclear localization of γ-catenin is frequent in AML. Significantly, γ-catenin expression was associated with ß-catenin stabilization and nuclear localization. Consistent with this, we found that ectopic γ-catenin expression promoted the stabilization and nuclear translocation of ß-catenin in leukemia cells. ß-Catenin knockdown demonstrated that both γ- and ß-catenin contribute to TCF-dependent transcription in leukemia cells. These data indicate that γ-catenin expression is a significant factor in the stabilization of ß-catenin in AML. We also show that although normal cells exclude nuclear translocation of both γ- and ß-catenin, this level of regulation is lost in the majority of AML patients and cell lines, which allow nuclear accumulation of these catenins and inappropriate TCF-dependent transcription.

Quality evaluation of clinical records of a group of general dental practitioners entering a quality assurance programme.

This paper discusses the importance of maintaining high quality clinical records. Evidence from studies carried out in the USA, Australia and Scandinavia shows that record keeping often falls well below accepted standards. Evidence of current standards in the UK, however, has tended to be anecdotal or circumstantial. An assessment was carried out on 47 general practitioners entering the quality assurance programme of a private capitation scheme. A sample of clinical records from each practitioner was analysed, and the presence or absence of key diagnostic and treatment planning entries were recorded. Overall, the quality of record keeping was poor, and in line with the findings of the other worldwide studies. Fundamental clinical entries that could impact on basic dental care provision were missing from many records. The frequency of recording for patients whose treatment was funded under NHS regulations was significantly worse than for patients whose treatment was privately funded.

Percutaneous radiologic drainage of pancreatic abscesses.

OBJECTIVE: The purpose of our study was to review and report the patient selection, techniques, and results of percutaneous drainage of pancreatic abscesses by retrospective review. MATERIALS AND METHODS: Fifty-nine patients (46 men and 13 women) with a mean age of 44 years old had 80 pancreatic abscesses that were drained percutaneously under radiologic guidance (CT, n = 77; sonography, n = 2; and fluoroscopy, n = 1). Abscesses had a wide spectrum of causes, with alcoholic pancreatitis being most common, trauma second most common, and gallstones third. Ten patients had undergone surgery for pancreatic necrosis or abscess. Patients with pancreatic pseudocysts, necrosis, or acute fluid collections were excluded from this study. RESULTS: Of the 59 patients, 51 (86%) were cured with percutaneous drainage and antibiotic therapy. Of the patients who were not cured with percutaneous drainage, seven required surgery and one underwent repeat percutaneous drainage. In the 59 patients, complications included non-life-threatening bleeding in three patients. Ten of 59 patients (17%) had fistulas that spontaneously formed into the gastrointestinal tract. The duration of catheterization ranged from 4 to 119 days, with a mean duration of 33 days. The rate of mortality at 30 days after completion of percutaneous drainage was 8% (5 of 59). CONCLUSION: Percutaneous drainage was an effective therapy for this defined group of patients with pancreatic abscesses. Factors leading to the relatively high success rate described in this study likely included selection of patients; catheters of adequate size, number, and location; careful follow-up with appropriate catheter manipulations; and an integrated, cooperative approach whereby surgeons were willing to permit drainage to effect its benefits, rather than operating prematurely.

New advances in electronic devices for hole detection.

Holes in surgical gloves are considered to be an important source of transmission of pathogens between surgeon and patient. Two new glove hole detectors have been devised to alert the surgeon to the presence of holes. These devices have been evaluated using six powder-free and seven powdered varieties of surgical gloves that were either dry or exposed to hydration. Eight of the 13 surgical gloves hydrated rapidly with water, altering their resistance to the conduction of electricity. Because the Barrier Integrity Monitor¿ only has a hydration monitor, 68 false positives occurred during the evaluation, indicating to the surgeon that he/she should change gloves unnecessarily because the glove had no hole. In contrast, the Surgic Alert Monitor¿ (SAM¿) had a hydration alarm as well as a glove hole detection alarm. During the 104 tests, the SAM¿ device showed no false positives. In the testing of five of the rapidly hydrating types of surgical gloves, the SAM¿ device could not reliably detect holes. On the basis of this study, the SAM¿ device, in conjunction with gloves that resist hydration, appeared to be a reliable hole detection monitor.

Treatment of lymphedema of the arms and legs with 5,6-benzo-[alpha]-pyrone.

BACKGROUND: Benzopyrones can reduce the volume of high-protein edema fluid by stimulating proteolysis. These compounds provide a method for removing excess protein and its consequent edema and reduce its clinical sequelae, such as chronic inflammation and secondary infections. METHODS: We conducted a randomized, double-blind, placebo-controlled, crossover trial of 5,6-benzo-[alpha]-pyrone in 31 patients with postmastectomy lymphedema of the arm and 21 patients with lymphedema of the leg of various causes (this agent, also known as 56 BaP, 1,2-benzopyrone, or coumarin, is not an anticoagulant). The patients received 400 mg of the active drug or placebo, each for six months. RESULTS: During the placebo period, lymphedema often worsened, especially in the arms. Measurements of limb volume showed that the active drug reduced the mean amount of edema fluid in the arms from 46 percent above normal to 26 percent above normal (P < 0.001) and the amount in the legs from 25 percent to 17 percent above normal (P < 0.001). The circumference of the arms was reduced from 17 percent to 13 percent above normal, and the circumference of the legs from 11 percent to 7 percent above normal (P < 0.001). The softness of the limb tissue was increased (P < 0.001), and elevated skin temperatures were reduced (P < 0.001). There were fewer attacks of secondary acute inflammation (P = 0.01). Bursting pains and feelings of hardness were decreased, as were feelings of tightness, tension, swelling, and heaviness; limb mobility also improved. The active drug was preferred to the placebo by 93 percent of the patients (P < 0.001). Side effects--mild nausea or diarrhea--occurred in seven patients taking the active drug. None withdrew from the trial, and the side effects disappeared after the first month of therapy. CONCLUSIONS: 5,6-Benzo-[alpha]-pyrone results in slow but safe reduction of lymphedema of the extremities.

The effect of trypsin inhibitor on the pancreas and small intestine of mice.

Pancreatic and intestinal growth rates were measured in mice fed on raw soya-bean flour (RSF) for up to 24 weeks. Control animals were fed on standard chow. The effects of RSF on the mouse pancreas resembled that seen in rats, showing hypertrophy with some hyperplasia. A marked increase in small intestinal weight was also found in mice fed on RSF but not in rats fed on this diet. Histological studies showed an increase in both villous and crypt thicknesses in the small intestine from these mice, and DNA, RNA and protein measurements indicated that the increase in intestinal weight was due to hypertrophy and hyperplasia of the mucosal layer. To determine whether the intestinal growth in mice fed on RSF was purely a response to the trypsin inhibitor (TI) component of the diet, pancreatic and intestinal growth rates were also determined in mice fed on the synthetic trypsin inhibitor camostate, at levels of 0.5 or 2 g/kg in rat chow, for periods of 1-8 weeks. Control animals were fed on standard chow. RSF and 0.5 g camostate/kg had similar trypsin inhibitor activities (measured against bovine trypsin), and both caused similar increases in pancreatic weight, DNA, RNA and protein content. However, 0.5 g camostate/kg did not affect small intestinal weight. Chow containing 2 g camostate/kg contained twice as much TI activity as the RSF diet but produced only a small increase in small intestinal weight at 2 and 8 weeks. This intestinal growth was significantly less than that seen with RSF. The present study shows that, in the mouse, RSF or a diet containing camostate in the appropriate dose produces pancreatic growth comparable to that seen in the rat. RSF also causes intestinal growth, but camostate-containing diets have little or no effect on the growth of the intestine.

Azaserine-induced pancreatic foci: detection, growth, labelling index and response to raw soya flour.

Atypical acinar cell foci were induced in the pancreases of rats by injection of azaserine. An incubation period of 6 weeks was sufficient for the detection of all glutathione S-transferase mu positive foci. In chow-fed rats, the labelling index of foci was 12-fold higher than normal pancreatic tissue. Feeding rats raw soya flour (RSF) for up to 20 weeks did not increase the number of foci per pancreas but did produce significant increases in labelling index and growth rate. In normal pancreatic tissue, the trophic response was complete after 4 weeks of RSF feeding. In foci, however, the trophic response to RSF was prolonged. Involution of normal pancreatic tissue was seen in rats fed RSF for 19 weeks and then switched to chow 1 week prior to death. No evidence for involution was seen in the foci of these animals, although a 40-fold reduction was seen in labelling index. The labelling index of these foci was reduced to the level seen in normal tissue of chow-fed rats. These results are consistent with increased cholecystokinin (CCK) responsiveness and CCK dependence in azaserine-induced pancreatic foci.

Complex physical therapy for the lymphoedematous arm.

Complex physical therapy was used in 78 patients with post-mastectomy lymphoedema (17 with grade 1 and 61 with grade 2). This involves: skin hygiene, a special lymphatic massage, compression bandaging and garments, and special exercises which supplement the massage. Two courses of treatment were given, lasting four weeks each, with a year between them. There was a highly significant decrease in the oedema in both grades, with more than 50% removed in the first course of treatment and 50% of the remainder in the second. There was even a small, but very significant decrease during the interval between the two courses.

A disposable emergency wound treatment kit.

Biomechanical studies in our laboratory have provided a scientific basis for selecting surgical gloves and instruments, drugs, and dressings for traumatic wound treatment. This armamentarium has been incorporated into a disposable emergency wound treatment kit for use in the emergency department. Its upper tray is for wound cleansing, while its lower tray is used for wound closure. A clinical evaluation of the kit by emergency physicians was very favorable because it saved time, eliminating the need to search for and assemble the gloves, instruments, drugs, and dressings for wound treatment.

The effect of fasting and cholecystokinin stimulation on normal and neoplastic tissue in the rat pancreas.

Pancreatic nodules were produced in rats by either feeding raw soya flour alone or by injection of azaserine plus raw soya flour feeding. The resulting nodules were studied to determine whether there was any functional difference between this tissue and the relatively normal internodular pancreas. Tissue DNA and trypsin content were significantly elevated in nodules compared to the adjacent tissue. With fasting, protein and enzyme content increased significantly and equally in both nodular and internodular tissues. RNA levels fell significantly and the decrease was more pronounced in nodular tissue. The responsiveness of the multinodular pancreas to cholecystokinin was examined by measuring pancreatic secretion basally and in response to cholecystokinin. Both the volume and protein content secreted by the multinodular pancreas were greatly elevated above control levels. When corrected for pancreatic weight, the difference remained significant and appeared to be due to increased basal secretion by the nodular pancreas. These studies demonstrate that azaserine-raw soya flour induced nodules are functionally efficient. Furthermore, the secretory response to cholecystokinin of these nodules is equal to or higher than that of normal tissue.

Glutathione S-transferase (mu class) as an early marker of azaserine-induced foci in the rat pancreas.

The alpha, mu and pi classes of glutathione S-transferase (GST) were evaluated as early immunocytochemical markers for the development of atypical foci within the pancreases of azaserine treated rats. Changes detected with haematoxylin and eosin (H&E) were compared with those detected by immunocytochemistry using antibodies raised against each class of GST. All foci detected with H&E staining were classified as acidophilic atypical acinar cell nodules (AACN), which have previously been reported in this model. All of these AACN overexpressed GST mu. However, 64% of foci detected with GST mu staining had not been identified as AACN during a prior examination with H&E. Re-evaluation of the H&E sections revealed that some of these foci showed subtle morphological changes which are indicative of AACN. In many cases, however, no morphological difference could be seen with H&E staining. We conclude that immunocytochemical staining for GST mu is a more reliable and sensitive method than H&E for detecting the early stages of azaserine-induced foci. Furthermore, we suggest that studies on the incidence and growth of these foci can be shortened considerably if GST mu staining is used in conjunction with H&E.

Changes in numbers of pancreatic acinar cell nuclei and in DNA content during raw soya flour feeding in mice.

Nuclei of pancreatic cells were isolated by trypsin-detergent digestion of fresh tissue and stained with propidium iodide, and nuclear DNA was measured by flow cytometry. Samples were isolated from mice fed either chow or raw soya flour (RSF) for periods ranging from 1 day to 48 weeks, beginning at 4 weeks of age. In chow-fed mice, the pancreas contained about 80% diploid (2N) and 20% tetraploid (4N) cells at the start of the study, but tetraploidy gradually increased to about 40% 2 weeks later (6 weeks of age) and remained at this level from that time onwards. Low levels of octaploid nuclei (8N) were also present in some animals after 2 weeks. In RSF-fed mice, about 20% tetraploid nuclei were also present for 1 and 2 days after starting RSF, but by 4 days tetraploidy had increased significantly to 40% and by 14 days had further increased to 50%. This level was significantly higher than that seen in chow-fed animals and was maintained for up to 48 weeks. Significantly higher numbers of octaploid nuclei were also present in the RSF-fed animals. In both chow- and RSF-fed mice, most cells were mononuclear, averaging 70% in chow-fed and 64% in RSF-fed animals. This difference was significant. This study shows that the mouse pancreas differs from the rat pancreas in the absence of a large population of binucleate acinar cells and the presence of considerable nuclear tetraploidy. Raw soya flour feeding leads to significant changes in these features, but in this species these changes do not appear to predispose to neoplasia.

Potentiation of azaserine by cholestyramine in the rat.

In the rat, when pancreatic growth is stimulated there is an increased incidence of spontaneous pancreatic neoplasms and marked potentiation of the pancreatic carcinogen azaserine. Since previous studies showed that cholestyramine caused pancreatic growth in this species we have now studied the effect of azaserine in rats fed soya flour diets containing cholestyramine. Two groups, each of eight rats, were fed either heated soya flour (HSF) or raw soya flour (RSF). Two further groups, each of 12 rats, received the same diets containing 2% cholestyramine (HSF + C, RSF + C). In each group, four rats received azaserine (30 mg/kg i.p.) and the remainder saline, weekly, for the first 5 weeks. Animals were killed after 24 weeks and pancreatic growth and the number and size of pancreatic neoplastic nodules was measured. RSF caused a significant increase in pancreatic weight, protein, RNA and DNA, compared with HSF and cholestyramine caused a further significant increase in pancreatic weight, protein and RNA but not DNA. Azaserine did not affect pancreatic growth. In azaserine-injected rats significantly more nodules were seen and the nodules were larger and the tumour burden greater in rats fed HSF + C than in rats fed HSF alone. However, the nodule count and other nodule parameters were not significantly different in RSF and RSF + C fed rats. It is concluded that 2% cholestyramine enhances pancreatic growth when added to soya flour diets and in rats fed HSF it potentiates the action of azaserine on the pancreas. It does not increase the potentiation of azaserine seen with RSF up to 24 weeks.

Cell proliferation in the exocrine pancreas during development.

This study examined the relative proliferation of the ductule cell compartment and the mononucleate and binucleate acinar cell populations in the developing pancreas in rats from 5 to 49 days of age. Proliferation of these cell types was assessed in the intact gland and in isolated acinar cells by autoradiography after in vivo labelling with tritiated thymidine at 5, 10, 17, 28, 35, 42 and 49 days of age. It was found that the acinar cell population was predominantly mononucleate at birth, but following weaning became progressively binucleate. At all times studied, DNA synthesis in mononucleate acinar cells was between 3- and 10-fold greater than in binucleate acinar cells. Ductule cell labelling was high relative to that seen in the adult from 5 to 17 days after birth, but after weaning duct cell labelling fell to levels seen in the adult. The results suggest that up to weaning acinus formation is derived from duct cell differentiation and mononucleate acinar cell proliferation, and that after weaning mononucleate acinar cells continue to replicate, either giving rise to binucleate acinar cells or continuing to divide as mononucleate cells. The mononucleate acinar cell thus appears to have the capacity to proliferate, while the binucleate acinar cell appears to be static and non-dividing.