IGF-1R pathway activation as putative biomarker for linsitinib therapy to revert tamoxifen resistance in ER-positive breast cancer

Preclinical studies indicate that activated IGF-1R can drive endocrine resistance in ER-positive (ER+) breast cancer, but its clinical relevance is unknown. We studied the effect of IGF-1R signaling on tamoxifen benefit in patients and we searched for approaches to overcome IGF-1R-mediated tamoxifen failure in cell lines. Primary tumor blocks from postmenopausal ER+ breast cancer patients randomized between adjuvant tamoxifen versus nil were recollected. Immunohistochemistry for IGF-1R, p-IGF-1R/InsR, p-ERα(Ser118), p-ERα(Ser167) and PI3K/MAPK pathway proteins was performed. Multivariate Cox models were employed to assess tamoxifen efficacy. The association between p-IGF-1R/InsR and PI3K/MAPK pathway activation in MCF-7 and T47D cells was analyzed with Western blots. Cell proliferation experiments were performed under various growth-stimulating and -inhibiting conditions. Patients with ER+, IGF-1R-positive breast cancer without p-IGF-1R/InsR staining (n = 242) had tamoxifen benefit (HR 0.41, p = 0.0038), while the results for p-IGF-1R/InsR-positive patients (n = 125) were not significant (HR 0.95, p = 0.3). High p-ERα(Ser118) or p-ERα(Ser167) expression was associated with less tamoxifen benefit. In MCF-7 cells, IGF-1R stimulation increased phosphorylation of PI3K/MAPK proteins and ERα(Ser167) regardless of IGF-1R overexpression. This could be abrogated by the dual IGF-1R/InsR inhibitor linsitinib, but not by the IGF-IR-selective antibody 1H7. In MCF-7 and T47D cells, stimulation of the IGF-1R/InsR pathway resulted in cell proliferation regardless of tamoxifen. Abrogation of cell growth was regained by addition of linsitinib. In conclusion, p-IGF-1R/InsR positivity in ER+ breast cancer is associated with reduced benefit from adjuvant tamoxifen in postmenopausal patients. In cell lines, stimulation rather than overexpression of IGF-1R is driving tamoxifen resistance to be abrogated by linsitinib.     

Biperiden and mepazine effectively inhibit MALT1 activity and tumor growth in pancreatic cancer

MALT1 is a key mediator of NF-κB signaling and a main driver of B-cell lymphomas. Remarkably, MALT1 is expressed in the majority of pancreatic ductal adenocarcinomas (PDACs) as well, but absent from normal exocrine pancreatic tissue. Following, MALT1 shows off to be a specific target in cancer cells of PDAC without affecting regular pancreatic cells. Therefore, we studied the impact of pharmacological MALT1 inhibition in pancreatic cancer and showed promising effects on tumor progression. Mepazine (Mep), a phenothiazine derivative, is a known potent MALT1 inhibitor. Newly, we described that biperiden (Bip) is a potent MALT1 inhibitor with even less pharmacological side effects. Thus, Bip is a promising drug leading to reduced proliferation and increased apoptosis in PDAC cells in vitro and in vivo. By compromising MALT1 activity, nuclear translocation of c-Rel is prevented. c-Rel is critical for NF-κB-dependent inhibition of apoptosis. Hence, off-label use of Bip or Mep represents a promising new therapeutic approach to PDAC treatment. Regularly, the Anticholinergicum Bip is used to treat neurological side effects of Phenothiazines, like extrapyramidal symptoms.     

The photobiology of the human circadian clock

In modern society, the widespread use of artificial light at night disrupts the suprachiasmatic nucleus (SCN), which serves as our central circadian clock. Existing models describe excitatory responses of the SCN to primarily blue light, but direct measures in humans are absent. The combination of state-of-the-art neuroimaging techniques and custom-made MRI compatible light-emitting diode devices allowed to directly measure the light response of the SCN. In contrast to the general expectation, we found that blood oxygen level–dependent (BOLD) functional MRI signals in the SCN were suppressed by light. The suppressions were observed not only in response to narrowband blue light (λ_max: 470 nm) but remarkably, also in response to green (λ_max: 515 nm) and orange (λ_max: 590 nm), but not to violet light (λ_max: 405 nm). The broadband sensitivity of the SCN implies that strategies on light exposure should be revised: enhancement of light levels during daytime is possible with wavelengths other than blue, while during nighttime, all colors are potentially disruptive.

Due to the Earth’s rotation around its axis, many organisms developed an internal clock to anticipate the predictable changes in the environment that occur every 24 h, including the daily light–dark cycle. In mammals, this clock is located in the suprachiasmatic nucleus (SCN), located in the hypothalamus directly above the optic chiasm (1, 2). The SCN receives information from the retina regarding ambient light levels via intrinsically photosensitive retinal ganglion cells (ipRGCs), thus synchronizing its internal clock to the external light–dark cycle. ipRGCs contain the photopigment melanopsin, which is maximally sensitive to blue light, with a peak response to 480-nm light (3, 4). In addition, ipRGCs also receive input from rod cells and cone cells (5–7). The three cone cell subtypes in the human retina respond maximally to 420-nm, 534-nm, and 563-nm light, while rod cells respond maximally to 498-nm light (8). In rodents, input from cone cells renders the SCN sensitive to a broad spectrum of wavelengths (9), while rod cells mediate the SCN’s sensitivity to low-intensity light (10, 11). Recently, these findings in rodents were proposed to translate to humans (12), suggesting that the human clock is not only sensitive to blue light, but may also be sensitive to other colors.In humans, circadian responses to light are generally measured indirectly (e.g., by measuring melatonin levels or 24-h behavioral rhythms). These indirect measures revealed that circadian responses to light in humans are most sensitive to blue light (13–16); however, green light has also been found to contribute to circadian phase shifting and changes in melatonin to a larger extent than would have been predicted based solely on the melanopsin response, suggesting that rods and/or cones may also provide functional input to the circadian system in humans (17). Despite this indirect evidence suggesting that several colors can affect the human circadian clock, this has never been measured directly due to technical limitations. Thus, current guidelines regarding the use of artificial light are based solely on the clock’s sensitivity to blue light. For example, blue light is usually filtered out in electronic screens during the night (18, 19), and blue-enriched light is used by night shift workers to optimize their body rhythm for achieving maximum performance (20–22).The ability to directly image the human SCN in vivo has been severely limited due to its small size and the relatively low spatial resolution provided by medical imaging devices. Previous functional MRI (fMRI) studies using 3-Tesla (3T) scanners were restricted to recording the “suprachiasmatic area,” which encompasses a large part of the hypothalamus and thus includes many other potentially light-sensitive nuclei (23–25). To overcome this limitation, we used a 7T MRI scanner, which can provide images with sufficiently high spatial resolution to image small brain nuclei (26) such as the SCN. Here, we applied colored light stimuli to healthy volunteers using a custom-designed MRI-compatible light-emitting diode (LED) device designed to stimulate specific photoreceptors while measuring SCN activity using fMRI. Using analytical approaches, we then identified the SCN’s response, the smallest brain nucleus that has so far been imaged. We found that the human SCN responds to a broad range of wavelengths (i.e., blue, green and orange light). Surprisingly, we also found that the blood oxygen level–dependent (BOLD) fMRI signal at the SCN is actually suppressed—not activated—by light.     

The effect of nightshift on emergency registrars' clinical skills

Objective: The effect of nightshift on ED staff performance is of clinical and risk‐management significance. Previous studies have demonstrated deterioration in psychomotor skills but the present study specifically assessed the impact of nightshift on clinical performance. Methods: The ED registrars in a tertiary hospital were enrolled in a prospective observational study and served as their own controls. During nightshift, subjects were presented simulated scenarios and tested with eight clinical questions developed to Fellowship examination standard. Matched scenarios and questions for the same subjects during dayshift served as controls. Two investigators, blinded to subject identity and the setting in which questions were attempted, independently collated answers. Results: Of 22 eligible subjects, all were recruited; four were excluded owing to incomplete data. A correlation of 0.99 was observed between the independent scoring investigators. Of a possible score of 17, the median result for nightshift was 9.5 (interquartile range: 8–11); corresponding value for dayshift was 12 (interquartile range: 10–13); P= 0.047. Conclusion: Nightshift effect on clinical performance is anecdotally well known. The present study quantifies such effects, specifically for the ED setting, and paves the way for focused research. The implications for clinical governance strategies are significant, as the fraternity embraces the mandate to maintain quality emergency care 24 h per day.     

Immunophenotypic features of MELF pattern invasion in endometrial adenocarcinoma: evidence for epithelial–mesenchymal transition

Aims:  Endometrial endometrioid adenocarcinomas (EEC) may show a distinctive morphological alteration characterized by the presence of microcystic, elongated and fragmented ('MELF') glands. These changes share features of epithelial–mesenchymal transition (EMT) in carcinomas arising at other sites. The aim was to compare the immunophenotypic profile of MELF-type epithelium with conventional glandular areas of EEC.
Methods and results:  Twenty-one EEC were stained immunohistochemically for cytokeratin (CK) AE1/AE3, CK7, vimentin, oestrogen receptor, progesterone receptor and E-cadherin. Conventional tumour glands usually showed preserved membranous E-cadherin immunoreactivity with peripheral accentuation of vimentin and hormone receptor expression. MELF-type invasion was characterized by strong CK7 expression, sometimes in contrast to adjacent unstained tumour glands. MELF areas were usually negative for hormone receptors and showed reduced E-cadherin expression.
Conclusions:  The expression of hormone receptors and intermediate filaments shows specific distribution patterns within EEC. MELF pattern invasion shows an altered immunophenotype compared with conventional glandular tumour areas. These findings suggest that MELF-type invasion represents a specific tumour alteration, and the reduction in hormone receptor and E-cadherin expression would be consistent with EMT. Immunohistochemical studies of EEC should consider micro anatomical variations in immunoreactivity, since these may be relevant to tumour invasion and progression.     

Characterization of the anti-HIV effects of native lactoferrin and other milk proteins and protein-derived peptides

In a search for natural proteins with anti-HIV activity, we screened a large set of purified proteins from bovine milk and peptide fragments thereof. Because several charged proteins and peptides are known to inhibit the process of virus entry, we selected proteins with an unusual charge composition or hydrophobicity profile. In contrast with some chemically modified (strongly negative) milk proteins, unmodified alpha(s2)-, beta- and kappa-casein, as well as several negatively and positively charged fragments thereof, did not show significant inhibition of virus replication. In fact, HIV-1 replication was elevated in the presence of beta-casein or amphiphilic fragments thereof. Bovine lactoferrin (bLF), a milk protein of 80 kDa, showed considerable inhibitory activity against HIV-1 with an IC50 of 0.4 microM. Modest inhibition was obtained with lactoferricin, a highly positively charged loop domain of bLF, indicating that other domains within the native bLF protein may also be required for inhibition. bLF blocked HIV-1 variants that use either the CXCR4 or the CCR5 coreceptor. In order to obtain further insight into the mechanism of action of this antiviral protein, we selected a bLF-resistant HIV-1 variant. The bLF-resistance phenotype is mediated by the viral envelope protein, which contains two interesting mutations that have previously been associated with an altered virus-host interaction and a modified receptor-coreceptor interaction. These results demonstrate that bLF targets the HIV-1 entry process.     

The Antiviral Protein Human Lactoferrin Is Distributed in the Body to Cytomegalovirus (CMV) Infection-Prone Cells and Tissues

Purpose. Lactoferrin has anti-Cytomegalovirus (CMV) and -HIV properties in vitro. However, the pharmacokinetic behavior of the 80-kD protein has not been well defined. We, therefore, assessed the plasma decay and body distribution of lactoferrin after intravenous administration to freely moving rats. Furthermore, the systemic availability of lactoferrin after intraperitoneal dosing was determined. Methods and Results. After intravenous injection, human lactoferrin (hLF) was rapidly cleared from the plasma, but higher doses resulted in prolonged plasma levels. Immunohistochemical analysis revealed a pronounced distribution of hLF to endothelial cells in the liver whereas diffuse staining in hepatocytes indicated the presence of considerable amounts in this large cell population. This endothelial association, which also was found in other organ/tissues, including blood vessels, was confirmed by in vitro cell-binding studies. In addition, leukocytes in plasma that were infiltrated in various organs showed binding of hLF. A small fraction of hLF was transported into the lymphatic system. Western blot analysis revealed that hLF, present in the various organs, mainly consisted of an 80-kD protein. After intraperitoneal administration, small amounts of 80-kD hLF distributed to the general circulation. The bioavailability was 0.6% but increased to 3.6% after multiple administrations. Conclusions. The affinity of hLF for endothelial cells and leukocytes, and its penetration into the lymphatic system, indicates that this protein reaches target cells and body compartments that are crucial for CMV and HIV replication. The ability to reach the blood compartment after intraperitoneal dosing offers opportunities for parenteral administration of the protein in future studies on its antiviral effects in vivo.     

The influence of charge clustering on the anti-HIV-1 activity and in vivo distribution of negatively charged albumins

The substitution of human serum albumin with negatively charged molecules, such as succinic acid (Suc-HSA) or aconitic acid (Aco-HSA), resulted in proteins with potent anti-HIV activities, by binding to viral gp120 (V3 loop). The aim of the present study was to investigate whether the distribution of negative charges on the albumin backbone influences the anti-HIV activity. Therefore, we prepared albumins with clusters of negatively charged groups by coupling of heparin. The effects of this substitution on anti-HIV activity, in vivo distribution and the protein structure as compared to random succinylation were assessed. In vitro studies indicated that HSA-modified with heparin 6 or 13 kD displayed anti-HIV activity (IC50=660 and 37 nM, respectively) and exhibited affinity for gp120-V3 loop, although the activity was lower than that of Suc-HSA. Combined derivatization of HSA with heparin 13 kD and aconitic acid groups resulted in significantly increased inhibitory actions (IC50=2.8 nM). Structural analysis showed that modification of HSA with heparin did not lead to extensive unfolding of the protein, meaning that these modified proteins were still globular in structure. In contrast, succinylation of HSA resulted in a highly randomly coiled conformation. Dynamic light scattering experiments revealed that, at neutral pH, the heparin fragments attached to the protein were wrapped around the molecule rather than sticking out into the solution. In conclusion, coupling of sufficient clustered negative charges, by coupling of Hep-fragments, on HSA resulted in a clear anti-HIV activity of the protein. Yet, random distribution of anionic groups in the albumin seemed more optimal for in vitro anti-HIV activity. The higher plasma and lymphatic concentrations of Hep-HSA compared to Suc-HSA seemed more favorable for an anti-HIV activity in vivo.