Protocol of the HISTOTHERM study: assessing the response to hyperthermia and hypofractionated radiotherapy in recurrent breast cancer.

Introduction: Breast cancer is globally the leading cancer in women, and despite the high 5-year survival rate the most frequent cause of cancer related deaths. Surgery, systemic therapy and radiotherapy are the three pillars of curative breast cancer treatment. However, locoregional recurrences frequently occur after initial treatment and are often challenging to treat, amongst others due to high doses of previous radiotherapy treatments. Radiotherapy can be combined with local hyperthermia to sensitize tumor cells to radiation and thereby significantly reduce the required radiation dose. Therefore, the combination treatment of mild local hyperthermia, i.e. locally heating of the tissue to 39-43°C, and re-irradiation with a reduced total dose is a relevant treatment option for previously irradiated patients. The mechanisms of this effect in the course of the therapy are to date not well understood and will be investigated in the HISTOTHERM study. Methods and analyses: Patients with local or (loco)regional recurrent breast cancer with macroscopic tumors are included in the study. Local tumor control is evaluated clinically and histologically during the course of a combination treatment of 60 minutes mild superficial hyperthermia (39 - 43°C) using water-filtered infrared A (wIRA) irradiation, immediately followed by hypofractionated re-irradiation with a total dose of 20-24 Gy, administered in weekly doses of 4 Gy. Tumor and tumor stroma biopsies as well as blood samples will be collected prior to treatment, during therapy (at a dose of 12 Gy) and in the follow-up to monitor therapy response. The treatment represents the standard operating procedure for hyperthermia plus re-irradiation. Various tissue and blood-based markers are analyzed. We aim at pinpointing key mechanisms and markers for therapy response which may help guiding treatment decisions in future. In addition, quality of life in the course of treatment will be assessed and survival data will be evaluated. Registration: The study is registered at the German Clinical Trials Register, Deutsches Register Klinischer Studien (DRKS00029221).

Krüppel-like factor 7 influences translation and pathways involved in ribosomal biogenesis in breast cancer.

BACKGROUND: Ribosomal biogenesis and ribosomal proteins have attracted attention in the context of tumor biology in recent years. Instead of being mere translational machineries, ribosomes might play an active role in tumor initiation and progression. Despite its importance, regulation of ribosomal biogenesis is still not completely understood. METHODS: Using Gene Set Enrichment Analysis of RNA sequencing and proteomical mass spectrometry data in breast cancer cells expressing Krüppel-like factor 7 (KLF7), we identified processes altered by this transcription factor. In silico analyses of a cohort of breast cancer patients in The Cancer Genome Atlas confirmed our finding. We further verified the role of KLF7 the identified ribosomal processes in in vitro assays of mammary carcinoma cell lines and analyses of breast cancer patients' tissue slices. RESULTS: We identified the transcription factor Krüppel-like factor 7 (KLF7) as a regulator of ribosomal biogenesis and translation in breast cancer cells and tissue. Highly significant overlapping processes related to ribosomal biogenesis were identified in proteomics and transcriptomics data and confirmed in patients' breast cancer RNA Seq data. Further, nucleoli, the sites of ribosomal biogenesis, were morphologically altered and quantitatively increased in KLF7-expressing cells. Pre-rRNA processing was identified as one potential process affected by KLF7. In addition, an increase in global translation independent from proliferation and transcription was observed upon exogenous KLF7 expression in vitro. Importantly, in a cohort of breast cancer patients, KLF7-expression levels correlated with aggressiveness of the intrinsic breast cancer subtype and tumor grading. Moreover, KLF7 correlated with nucleolar characteristics in human breast tumor tissue, indicating a role for KLF7 in ribosomal biogenesis. CONCLUSION: In mammary carcinoma, KLF7 is involved in ribosomal biogenesis. Alterations of ribosomal biogenesis has far reaching quantitative and qualitative implications for the proteome of the cancer cells. This might influence the aggressiveness of cancer cells.

Prognostic Impact of Tumor Budding in Intrahepatic Cholangiocellular Carcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) represents an aggressive carcinoma with a dismal prognosis. For resection specimens, histopathological prognosticators are limited to standard AJCC parameters. Tumor budding (TB), a quantitative leviable parameter for tumor cell separation and infiltration is a promising prognostic factor for several cancers. This retrospective study investigated the prognostic impact of tumor budding in ICC, using a semi-automated approach. METHOD: From the Memorial Sloan-Kettering Cancer Center pathology archives, tissue specimens from ICC patients were HE stained and digitized. Tumor budding was analyzed according to the International Tumor Budding Consensus Conference 2016 via QuPath in ten 0.785 mm² vision fields within the tumor center and the tumor-host interface. Within each field, automated QuPath cell detection was conducted and manually reviewed. Tumor budding was correlated with clinico-pathological parameters including AJCC 8th edition classification, hepatitis status, age, ethnicity, treatment, sex, patient overall (OS) and recurrence free survival (RFS) via uni- and multivariate analyses. RESULTS: From 89 patients, 1780 Vision fields comprising 6006 tumor buds were analyzed and correlated with patients' OS and RFS. The median value for tumor budding in tumor budding hot spots was five within the tumor-host interface and six within the tumor center. Tumor budding correlated significantly with patient OS and RFS in uni- and multivariate analyses (p<0.001). CONCLUSION: Our data supports tumor budding, assessed using a digitally enhanced technique, as an independent prognosticator in ICCs for patient's OS and RFS.

Early assessment of circulating tumor DNA after curative-intent resection predicts tumor recurrence in early-stage and locally advanced non-small-cell lung cancer.

Circulating tumor DNA (ctDNA) has demonstrated great potential as a noninvasive biomarker to assess minimal residual disease (MRD) and profile tumor genotypes in patients with non-small-cell lung cancer (NSCLC). However, little is known about its dynamics during and after tumor resection, or its potential for predicting clinical outcomes. Here, we applied a targeted-capture high-throughput sequencing approach to profile ctDNA at various disease milestones and assessed its predictive value in patients with early-stage and locally advanced NSCLC. We prospectively enrolled 33 consecutive patients with stage IA to IIIB NSCLC undergoing curative-intent tumor resection (median follow-up: 26.2 months). From 21 patients, we serially collected 96 plasma samples before surgery, during surgery, 1-2 weeks postsurgery, and during follow-up. Deep next-generation sequencing using unique molecular identifiers was performed to identify and quantify tumor-specific mutations in ctDNA. Twelve patients (57%) had detectable mutations in ctDNA before tumor resection. Both ctDNA detection rates and ctDNA concentrations were significantly higher in plasma obtained during surgery compared with presurgical specimens (57% versus 19% ctDNA detection rate, and 12.47 versus 6.64 ng·mL-1 , respectively). Four patients (19%) remained ctDNA-positive at 1-2 weeks after surgery, with all of them (100%) experiencing disease progression at later time points. In contrast, only 4 out of 12 ctDNA-negative patients (33%) after surgery experienced relapse during follow-up. Positive ctDNA in early postoperative plasma samples was associated with shorter progression-free survival (P = 0.013) and overall survival (P = 0.004). Our findings suggest that, in early-stage and locally advanced NSCLC, intraoperative plasma sampling results in high ctDNA detection rates and that ctDNA positivity early after resection identifies patients at risk for relapse.

Golgi-Resident Gαo Promotes Protrusive Membrane Dynamics.

To form protrusions like neurites, cells must coordinate their induction and growth. The first requires cytoskeletal rearrangements at the plasma membrane (PM), the second requires directed material delivery from cell's insides. We find that the Gαo-subunit of heterotrimeric G proteins localizes dually to PM and Golgi across phyla and cell types. The PM pool of Gαo induces, and the Golgi pool feeds, the growing protrusions by stimulated trafficking. Golgi-residing KDELR binds and activates monomeric Gαo, atypically for G protein-coupled receptors that normally act on heterotrimeric G proteins. Through multidimensional screenings identifying > 250 Gαo interactors, we pinpoint several basic cellular activities, including vesicular trafficking, as being regulated by Gαo. We further find small Golgi-residing GTPases Rab1 and Rab3 as direct effectors of Gαo. This KDELR → Gαo → Rab1/3 signaling axis is conserved from insects to mammals and controls material delivery from Golgi to PM in various cells and tissues.

Mode of interaction of the Gαo subunit of heterotrimeric G proteins with the GoLoco1 motif of Drosophila Pins is determined by guanine nucleotides.

Drosophila GoLoco motif-containing protein Pins is unusual in its highly efficient interaction with both GDP- and the GTP-loaded forms of the α-subunit of the heterotrimeric Go protein. We analysed the interactions of Gαo in its two nucleotide forms with GoLoco1-the first of the three GoLoco domains of Pins-and the possible structures of the resulting complexes, through combination of conventional fluorescence and FRET measurements as well as through molecular modelling. Our data suggest that the orientation of the GoLoco1 motif on Gαo significantly differs between the two nucleotide states of the latter. In other words, a rotation of the GoLoco1 peptide in respect with Gαo must accompany the nucleotide exchange in Gαo. The sterical hindrance requiring such a rotation probably contributes to the guanine nucleotide exchange inhibitor activity of GoLoco1 and Pins as a whole. Our data have important implications for the mechanisms of Pins regulation in the process of asymmetric cell divisions.

Lack of evidence of the interaction of the Aß peptide with the Wnt signaling cascade in Drosophila models of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the leading form of dementia worldwide. The Aß-peptide is believed to be the major pathogenic compound of the disease. Since several years it is hypothesized that Aß impacts the Wnt signaling cascade and therefore activation of this signaling pathway is proposed to rescue the neurotoxic effect of Aß. FINDINGS: Expression of the human Aß42 in the Drosophila nervous system leads to a drastically shortened life span. We found that the action of Aß42 specifically in the glutamatergic motoneurons is responsible for the reduced survival. However, we find that the morphology of the glutamatergic larval neuromuscular junctions, which are widely used as the model for mammalian central nervous system synapses, is not affected by Aß42 expression. We furthermore demonstrate that genetic activation of the Wnt signal transduction pathway in the nervous system is not able to rescue the shortened life span or a rough eye phenotype in Drosophila. CONCLUSIONS: Our data confirm that the life span is a useful readout of Aß42 induced neurotoxicity in Drosophila; the neuromuscular junction seems however not to be an appropriate model to study AD in flies. Additionally, our results challenge the hypothesis that Wnt signaling might be implicated in Aß42 toxicity and might serve as a drug target against AD.

Heterotrimeric Go protein links Wnt-Frizzled signaling with ankyrins to regulate the neuronal microtubule cytoskeleton.

Drosophila neuromuscular junctions (NMJs) represent a powerful model system with which to study glutamatergic synapse formation and remodeling. Several proteins have been implicated in these processes, including components of canonical Wingless (Drosophila Wnt1) signaling and the giant isoforms of the membrane-cytoskeleton linker Ankyrin 2, but possible interconnections and cooperation between these proteins were unknown. Here, we demonstrate that the heterotrimeric G protein Go functions as a transducer of Wingless-Frizzled 2 signaling in the synapse. We identify Ankyrin 2 as a target of Go signaling required for NMJ formation. Moreover, the Go-ankyrin interaction is conserved in the mammalian neurite outgrowth pathway. Without ankyrins, a major switch in the Go-induced neuronal cytoskeleton program is observed, from microtubule-dependent neurite outgrowth to actin-dependent lamellopodial induction. These findings describe a novel mechanism regulating the microtubule cytoskeleton in the nervous system. Our work in Drosophila and mammalian cells suggests that this mechanism might be generally applicable in nervous system development and function.

Wnt secretion and gradient formation.

Concentration gradients formed by the lipid-modified morphogens of the Wnt family are known for their pivotal roles during embryogenesis and adult tissue homeostasis. Wnt morphogens are also implicated in a variety of human diseases, especially cancer. Therefore, the signaling cascades triggered by Wnts have received considerable attention during recent decades. However, how Wnts are secreted and how concentration gradients are formed remains poorly understood. The use of model organisms such as Drosophila melanogaster has provided important advances in this area. For instance, we have previously shown that the lipid raft-associated reggie/flotillin proteins influence Wnt secretion and spreading in Drosophila. Our work supports the notion that producing cells secrete Wnt molecules in at least two pools: a poorly diffusible one and a reggie/flotillin-dependent highly diffusible pool which allows morphogen spreading over long distances away from its source of production. Here we revise the current views of Wnt secretion and spreading, and propose two models for the role of the reggie/flotillin proteins in these processes: (i) reggies/flotillins regulate the basolateral endocytosis of the poorly diffusible, membrane-bound Wnt pool, which is then sorted and secreted to apical compartments for long-range diffusion, and (ii) lipid rafts organized by reggies/flotillins serve as "dating points" where extracellular Wnt transiently interacts with lipoprotein receptors to allow its capture and further spreading via lipoprotein particles. We further discuss these processes in the context of human breast cancer. A better understanding of these phenomena may be relevant for identification of novel drug targets and therapeutic strategies.