Repurposing proteasome inhibitors for improved treatment of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is associated with poor prognosis and limited treatment options due to the lack of important receptors (estrogen receptor [ER], progesterone receptor [PR], and human epidermal growth factor receptor 2 [HER2]) used for targeted therapy. However, high-throughput in vitro drug screening of cell lines is a powerful tool for identifying effective drugs for a disease. Here, we determine the intrinsic chemosensitivity of TNBC cell lines to proteasome inhibitors (PIs), thereby identifying potentially potent 2-drug combinations for TNBC. Eight TNBC cell lines (BT-549, CAL-148, HCC1806, HCC38, HCC70, MDA-MB-436, MDA-MB-453, and MDA-MB-468) and two controls (MCF-10A and MCF-7) were first exposed to 18 drugs (11 PIs and 7 clinically relevant chemotherapeutic agents) as monotherapy, followed by prediction of potent 2-drug combinations using the IDACombo pipeline. The synergistic effects of the 2-drug combinations were evaluated with SynergyFinder in four TNBC cell lines (CAL-148, HCC1806, HCC38, and MDA-MB-468) and three controls (BT-474, MCF-7, and T47D) in vitro, followed by further evaluation of tumor regression in zebrafish tumor models established using HCC1806 and MCF-7 cells. Monotherapy identified nine effective drugs (bortezomib, carfilzomib, cisplatin, delanzomib, docetaxel, epoxomicin, MLN-2238, MLN-9708, and nedaplatin) across all cell lines. PIs (e.g., bortezomib, delanzomib, and epoxomicin) were highly potent drugs in TNBC cells, of which bortezomib and delanzomib inhibited the chymotrypsin-like activity of the 20 S proteasome by 100% at 10 µM. Moreover, several potent 2-drug combinations (e.g., bortezomib+nedaplatin and epoxomicin+epirubicin) that killed virtually 100% of cells were also identified. Although HCC1806- and MCF-7-derived xenografts treated with bortezomib+nedaplatin and carboplatin+paclitaxel were smaller, HCC1806 cells frequently metastasized to the trunk region. Taken together, we show that PIs used in combination with platinum agents or topoisomerase inhibitors exhibit increased efficiency with almost 100% inhibition in TNBC cell lines, indicating that PIs are therefore promising compounds to use as combination therapy for TNBC.

Analysis of radioactivity in commercially available products aiming to improve health and wellness.

There are products available on the online market that are claim to contain unique 'energies' that can improve health and wellness by eliminating toxins and pains and energising food and drinking water. We investigated these products by alpha and gamma spectrometry, and the analysis showed that they contained a few to hundreds of kilobecquerels per kilogram of naturally occurring radionuclides from the 232Th and 238U series. The committed effective dose for an adult drinking water that had been in contact with these products just once was estimated to 12 nSv. Considering a worst-case scenario for the workers inhaling the radioactive substance, 1 d of work would result in an effective dose of 0.39 mSv. The product descriptions do not mention the radionuclide content, and concerns are raised for the consumers and workers exposed to these products with no knowledge of the radioactive content.

Recombinant α1-Microglobulin (rA1M) Protects against Hematopoietic and Renal Toxicity, Alone and in Combination with Amino Acids, in a 177Lu-DOTATATE Mouse Radiation Model.

177Lu-DOTATATE peptide receptor radionuclide therapy (PRRT) is used clinically to treat metastasized or unresectable neuroendocrine tumors (NETs). Although 177Lu-DOTATATE is mostly well tolerated in patients, bone marrow suppression and long-term renal toxicity are still side effects that should be considered. Amino acids are often used to minimize renal radiotoxicity, however, they are associated with nausea and vomiting in patients. α1-microglobulin (A1M) is an antioxidant with heme- and radical-scavenging abilities. A recombinant form (rA1M) has previously been shown to be renoprotective in preclinical models, including in PRRT-induced kidney damage. Here, we further investigated rA1M's renal protective effect in a mouse 177Lu-DOTATATE model in terms of administration route and dosing regimen and as a combined therapy with amino acids (Vamin). Moreover, we investigated the protective effect of rA1M on peripheral blood and bone marrow cells, as well as circulatory biomarkers. Intravenous (i.v.) administration of rA1M reduced albuminuria levels and circulatory levels of the oxidative stress-related protein fibroblast growth factor-21 (FGF-21). Dual injections of rA1M (i.e., at 0 and 24 h post-177Lu-DOTATATE administration) preserved bone marrow cellularity and peripheral blood reticulocytes. Administration of Vamin, alone or in combination with rA1M, did not show any protection of bone marrow cellularity or peripheral reticulocytes. In conclusion, this study suggests that rA1M, administered i.v. for two consecutive days in conjunction with 177Lu-DOTATATE, may reduce hematopoietic and kidney toxicity during PRRT with 177Lu-DOTATATE.

Co-administration with A1M does not influence apoptotic response of 177Lu-octreotate in GOT1 neuroendocrine tumors.

Recombinant α1-microglobulin (A1M) is a proposed radioprotector during 177Lu-octreotate therapy of neuroendocrine tumors (NETs). To ensure a maintained therapeutic effect, we previously demonstrated that A1M does not affect the 177Lu-octreotate induced decrease in GOT1 tumor volume. However, the underlying biological events of these findings are still unknown. The aim of this work was to examine the regulation of apoptosis-related genes in GOT1 tumors short-time after i.v. administration of 177Lu-octreotate with and without A1M or A1M alone. Human GOT1 tumor-bearing mice received 30 MBq 177Lu-octreotate or 5 mg/kg A1M or co-treatment with both. Animals were sacrificed after 1 or 7 days. Gene expression analysis of apoptosis-related genes in GOT1 tissue was performed with RT-PCR. In general, similar expression patterns of pro- and anti-apoptotic genes were found after 177Lu-octreotate exposure with or without co-administration of A1M. The highest regulated genes in both irradiated groups compared to untreated controls were FAS and TNFSFRS10B. Administration of A1M alone only resulted in significantly regulated genes after 7 days. Co-administration of A1M did not negatively affect the transcriptional apoptotic response of 177Lu-octreotate in GOT1 tumors.

Pan-cancer analysis of genomic and transcriptomic data reveals the prognostic relevance of human proteasome genes in different cancer types.

BACKGROUND: The human proteasome gene family (PSM) consists of 49 genes that play a crucial role in cancer proteostasis. However, little is known about the effect of PSM gene expression and genetic alterations on clinical outcome in different cancer forms. METHODS: Here, we performed a comprehensive pan-cancer analysis of genetic alterations in PSM genes and the subsequent prognostic value of PSM expression using data from The Cancer Genome Atlas (TCGA) containing over 10,000 samples representing up to 33 different cancer types. External validation was performed using a breast cancer cohort and KM plotter with four cancer types. RESULTS: The PSM genetic alteration frequency was high in certain cancer types (e.g. 67%; esophageal adenocarcinoma), with DNA amplification being most common. Compared with normal tissue, most PSM genes were predominantly overexpressed in cancer. Survival analysis also established a relationship with PSM gene expression and adverse clinical outcome, where PSMA1 and PSMD11 expression were linked to more unfavorable prognosis in ≥ 30% of cancer types for both overall survival (OS) and relapse-free interval (PFI). Interestingly, PSMB5 gene expression was associated with OS (36%) and PFI (27%), and OS for PSMD2 (42%), especially when overexpressed. CONCLUSION: These findings indicate that several PSM genes may potentially be prognostic biomarkers and novel therapeutic targets for different cancer forms.

Age and sex effects across the blood proteome after ionizing radiation exposure can bias biomarker screening and risk assessment.

Molecular biomarkers of ionizing radiation (IR) exposure are a promising new tool in various disciplines: they can give necessary information for adaptive treatment planning in cancer radiotherapy, enable risk projection for radiation-induced survivorship diseases, or facilitate triage and intervention in radiation hazard events. However, radiation biomarker discovery has not yet resolved the most basic features of personalized medicine: age and sex. To overcome this critical bias in biomarker identification, we quantitated age and sex effects and assessed their relevance in the radiation response across the blood proteome. We used high-throughput mass spectrometry on blood plasma collected 24 h after 0.5 Gy total body irradiation (15 MV nominal photon energy) from male and female C57BL/6 N mice at juvenile (7-weeks-old) or adult (18-weeks-old) age. We also assessed sex and strain effects using juvenile male and female BALB/c nude mice. We showed that age and sex created significant effects in the proteomic response regarding both extent and functional quality of IR-induced responses. Furthermore, we found that age and sex effects appeared non-linear and were often end-point specific. Overall, age contributed more to differences in the proteomic response than sex, most notably in immune responses, oxidative stress, and apoptotic cell death. Interestingly, sex effects were pronounced for DNA damage and repair pathways and associated cellular outcome (pro-survival vs. pro-apoptotic). Only one protein (AHSP) was identified as a potential general biomarker candidate across age and sex, while GMNN, REG3B, and SNCA indicated some response similarity across age. This low yield advocated that unisex or uniage biomarker screening approaches are not feasible. In conclusion, age- and sex-specific screening approaches should be implemented as standard protocol to ensure robustness and diagnostic power of biomarker candidates. Bias-free molecular biomarkers are a necessary progression towards personalized medicine and integral for advanced adaptive cancer radiotherapy and risk assessment.

Natural radioactivity and element characterization in pit lakes in Northern Sweden.

Northern Sweden has been the object of intense metal mining in the last decades producing several water-filled open-pits, or pit lakes. Most of these pit lakes have been limed to maintain a good water quality and to prevent generation of acidic water that could leach the exposed rocks and release metals into water. The aim of this work was to examine the concentration of stable elements and naturally occurring radionuclides in water and sediment samples from pit lakes originating from non-uranium mining activities in Northern Sweden. Surface water and surface sediments were collected from 27 pit lakes in Northern Sweden. Water quality parameters, concentration of stable elements and radionuclides were measured by a water probe, ICP-MS and XRF, and alpha and gamma spectrometry, respectively. Furthermore, a multivariate statistical analysis (PCA) was performed on the water samples and sediments. In general, the quality of the surface water was good, but some lakes had low pH values (2.5-5.7), and high concentrations of Fe (up to 200 mg/L) and other metals (e.g. Zn, Cu). When relating the metal concentrations in sediments in pit lakes with the concentration found in natural lakes, some sites had relatively high levels of Cu, As, Cr and Pb. The activity concentration of 210Po, and U and Th isotopes in water and sediment samples were at environmental levels, as was the ambient dose equivalent rate at these sites (range 0.08-0.14 µSv/h).

Age-related long-term response in rat thyroid tissue and plasma after internal low dose exposure to 131I.

131I is used clinically for therapy, and may be released during nuclear accidents. After the Chernobyl accident papillary thyroid carcinoma incidence increased in children, but not adults. The aims of this study were to compare 131I irradiation-dependent differences in RNA and protein expression in the thyroid and plasma of young and adult rats, and identify potential age-dependent biomarkers for 131I exposure. Twelve young (5 weeks) and twelve adult Sprague Dawley rats (17 weeks) were i.v. injected with 50 kBq 131I (absorbed dose to thyroid = 0.1 Gy), and sixteen unexposed age-matched rats were used as controls. The rats were killed 3-9 months after administration. Microarray analysis was performed using RNA from thyroid samples, while LC-MS/MS analysis was performed on proteins extracted from thyroid tissue and plasma. Canonical pathways, biological functions and upstream regulators were analysed for the identified transcripts and proteins. Distinct age-dependent differences in gene and protein expression were observed. Novel biomarkers for thyroid 131I exposure were identified: (PTH), age-dependent dose response (CA1, FTL1, PVALB (youngsters) and HSPB6 (adults)), thyroid function (Vegfb (adults)). Further validation using clinical samples are needed to explore the role of the identified biomarkers.

Hyperfractionated Treatment with 177Lu-Octreotate Increases Tumor Response in Human Small-Intestine Neuroendocrine GOT1 Tumor Model.

Radionuclide treatment of patients with neuroendocrine tumors has advanced in the last decades with favorable results using 177Lu-octreotate. However, the gap between the high cure rate in animal studies vs. patient studies indicates a potential to increase the curation of patients. The aim of this study was to investigate the tumor response for different fractionation schemes with 177Lu-octreotate. BALB/c mice bearing a human small-intestine neuroendocrine GOT1 tumor were either mock treated with saline or injected intravenously with a total of 30-120 MBq of 177Lu-octreotate: 1 × 30, 2 × 15, 1 × 60, 2 × 30, 1 × 120, 2 × 60, or 3 × 40 MBq. The tumor volume was measured twice per week until the end of the experiment. The mean tumor volume for mice that received 2 × 15 = 30 and 1 × 30 MBq 177Lu-octreotate was reduced by 61% and 52%, respectively. The mean tumor volume was reduced by 91% and 44% for mice that received 2 × 30 = 60 and 1 × 60 MBq 177Lu-octreotate, respectively. After 120 MBq 177Lu-octreotate, given as 1-3 fractions, the mean tumor volume was reduced by 91-97%. Multiple fractions resulted in delayed regrowth and prolonged overall survival by 20-25% for the 120 MBq groups and by 45% for lower total activities, relative to one fraction. The results indicate that fractionation and hyperfractionation of 177Lu-octreotate are beneficial for tumor reduction and prolongs the time to regrowth.

VERDICT MRI for radiation treatment response assessment in neuroendocrine tumors.

Noninvasive methods to study changes in tumor microstructure enable early assessment of treatment response and thus facilitate personalized treatment. The aim of this study was to evaluate the diffusion MRI model, Vascular, Extracellular and Restricted Diffusion for Cytometry in Tumors (VERDICT), for early response assessment to external radiation treatment and to compare the results with those of more studied sets of parameters derived from diffusion-weighted MRI data. Mice xenografted with human small intestine tumors were treated with external radiation treatment, and diffusion MRI experiments were performed on the day before and up to 2 weeks after treatment. The diffusion models VERDICT, ADC, IVIM, and DKI were fitted to MRI data, and the treatment response of each tumor was calculated based on pretreatment tumor growth and post-treatment tumor volume regression. Linear regression and correlation analysis were used to evaluate each model and their respective parameters for explaining the treatment response. VERDICT analysis showed significant changes from day -1 to day 3 for the intracellular and extracellular volume fraction, as well as the cell radius index (p < 0.05; Wilcoxon signed-rank test). The strongest correlation between the diffusion model parameters and the tumor treatment response was seen for the ADC, kurtosis-corrected diffusion coefficient, and intracellular volume fraction on day 3 (τ = 0.47, 0.52, and -0.49, respectively, p < 0.05; Kendall rank correlation coefficient). Of all the tested models, VERDICT held the strongest explanatory value for the tumor treatment response on day 3 (R2  = 0.75, p < 0.01; linear regression). In conclusion, VERDICT has potential for early assessment of external radiation treatment and may provide further insights into the underlying biological effects of radiation on tumor tissue. In addition, the results suggest that the time window for assessment of treatment response using dMRI may be narrow.

Correction: Long-term transcriptomic and proteomic effects in Sprague Dawley rat thyroid and plasma after internal low dose 131I exposure.

[This corrects the article DOI: 10.1371/journal.pone.0244098.].

Neurochemical properties measured by 1H magnetic resonance spectroscopy may predict cognitive behaviour therapy outcome in paediatric OCD: a pilot study.

To identify neurochemical factors measured pre-treatment that may predict cognitive behavioural treatment (CBT) outcome, aiming at understanding possible causes of poor CBT response. 1H magnetic resonance spectroscopy was used before treatment with CBT in treatment naïve 11-18 year-old patients with moderate-severe OCD. Diagnoses and assessment of OCD severity were based on semi-structured interviews. Linear mixed effects models were used to analyse the association between metabolite level and treatment outcome. Worse CBT outcome was associated with higher concentration of glutamine and glutamate combined (Glx) in middle cingulate cortex (MCC) (F = + 3.35, p = 0.004) and of N-acetylaspartate and N-acetylaspartylglutamate combined (tNAA) (F = + 2.59, p = 0.019). Also, we noted a tendency towards higher thalamic Glx concentration (F = + 1.91, p = 0.077) to be associated with worse CBT outcome. In general, the findings of the current pilot study are compatible with the hypothesis of an overweight of excitatory to inhibitory factors in brain circuits driving goal-directed behaviours (GDB). Higher MCC Glx and tNAA may be involved in the selection of GDB. A more detailed understanding of how these brain areas function in health and illness is needed.

Neuroblastoma xenograft models demonstrate the therapeutic potential of 177Lu-octreotate.

BACKGROUND: Neuroblastoma (NB) is one of the most frequently diagnosed tumors in infants. NB is a neuroendocrine tumor type with various characteristics and features, and with diverse outcome. The most malignant NBs have a 5-year survival rate of only 40-50%, indicating the need for novel and improved treatment options. 177Lu-octreotate is routinely administered for treatment of neuroendocrine tumors overexpressing somatostatin receptors (SSTR). The aim of this study was to examine the biodistribution of 177Lu-octreotate in mice bearing aggressive human NB cell lines, in order to evaluate the potential usefulness of 177Lu-octreotate for treatment of NB. METHODS: BALB/c nude mice bearing CLB-BAR, CLB-GE or IMR-32 tumor xenografts (n = 5-7/group) were i.v. injected with 0.15 MBq, 1.5 MBq or 15 MBq 177Lu-octreotate and sacrificed 1 h, 24 h, 48 h and 168 h after administration. The radioactivity concentration was determined for collected tissue samples, tumor-to-normal-tissue activity concentration ratios (T/N) and mean absorbed dose for each tissue were calculated. Immunohistochemical (IHC) staining for SSTR1-5, and Ki67 were carried out for tumor xenografts from the three cell lines. RESULTS: High 177Lu concentration levels and T/N values were observed in all NB tumors, with the highest for CLB-GE tumor xenografts (72%IA/g 24 h p.i.; 1.5 MBq 177Lu-octreotate). The mean absorbed dose to the tumor was 6.8 Gy, 54 Gy and 29 Gy for CLB-BAR, CLB-GE and IMR-32, respectively, p.i. of 15 MBq 177Lu-octreotate. Receptor saturation was clearly observed in CLB-BAR, resulting in higher concentration levels in the tumor when lower activity levels where administered. IHC staining demonstrated highest expression of SSTR2 in CLB-GE, followed by CLB-BAR and IMR-32. CONCLUSION: T/N values for all three human NB tumor xenograft types investigated were high relative to previously investigated neuroendocrine tumor types. The results indicate a clear potential of 177Lu-octreotate as a therapeutic alternative for metastatic NB.

Genetic alterations associated with multiple primary malignancies.

Breast cancer (BC) patients are frequently at risk of developing other malignancies following treatment. Although studies have been conducted to elucidate the etiology of multiple primary malignancies (MPM) after a BC diagnosis, few studies have investigated other previously diagnosed primary malignancies (OPPM) before BC. Here, genome-wide profiling was used to identify potential driver DNA copy number alterations and somatic mutations that promote the development of MPMs. To compare the genomic profiles for two primary tumors (BC and OPPM) from the same patient, tumor pairs from 26 young women (≤50 years) diagnosed with one or more primary malignancies before breast cancer were analyzed. Malignant melanoma was the most frequent OPPM, followed by gynecologic- and hematologic malignancies. However, significantly more genetic alterations were detected in BC compared to the OPPM. BC also showed more genetic similarity as a group than the tumor pairs. Clonality testing showed that genetic alterations on chromosomes 1, 3, 16, and 19 were concordant in both tumors in 13 patients. TP53 mutations were also found to be prevalent in BC, MM, and HM. Although all samples were classified as genetically unstable, chromothripsis-like patterns were primarily observed in BC. Taken together, few recurrent genetic alterations were identified in both tumor pairs that can explain the development of MPMs in the same patient. However, larger studies are warranted to further investigate key driver mutations associated with MPMs.

Effects of different mean arterial pressure targets on plasma volume, ANP and glycocalyx-A randomized trial.

BACKGROUND: Arterial haematocrit (Hct) has been shown to decrease after anaesthesia induction, most probably because of an increased plasma volume (PV). The primary objective was to quantify change in PV if mean arterial pressure (MAP) was kept at baseline level or allowed to decrease to 60 mm Hg. Our secondary objective was to evaluate underlying mechanisms of this response. METHODS: Twenty-four coronary artery bypass patients were randomized to a higher (90 mm Hg, intervention group) or lower (60 mm Hg, control group) MAP by titration of norepinephrine. During the experimental procedure, no fluids were administered. Baseline PV was measured by 125 I-albumin and the change in PV was calculated from the change in Hct. Changes in MAP, plasma 125 I-albumin, colloid osmotic pressure, albumin, Mid Regional-pro Atrial Natriuretic Peptide (MR-proANP) and endothelial glycocalyx components were measured from baseline to 50 minutes after anaesthesia induction. RESULTS: The MAP during the trial was 93 ± 9 mm Hg in the intervention group and 62 ± 5 mm Hg in the control group. PV increased with up to 420 ± 180 mL in the control group and 45 ± 130 mL in the intervention group (P < .001). Albumin and colloid osmotic pressure decreased significantly more in the control group. MR-proANP increased in the control group but no shedding of the glycocalyx layer was detected in either of the groups. CONCLUSION: Allowing mean arterial pressure to fall to 60 mm Hg during anaesthesia induction, increases the plasma volume due to reabsorption of interstitial water, with no ANP-induced degradation of the endothelial glycocalyx.

Long-term transcriptomic and proteomic effects in Sprague Dawley rat thyroid and plasma after internal low dose 131I exposure.

BACKGROUND: Radioiodide (131I) is commonly used to treat thyroid cancer and hyperthyroidis.131I released during nuclear accidents, have resulted in increased incidence of thyroid cancer in children. Therefore, a better understanding of underlying cellular mechanisms behind 131I exposure is of great clinical and radiation protection interest. The aim of this work was to study the long-term dose-related effects of 131I exposure in thyroid tissue and plasma in young rats and identify potential biomarkers. MATERIALS AND METHODS: Male Sprague Dawley rats (5-week-old) were i.v. injected with 0.5, 5.0, 50 or 500 kBq 131I (Dthyroid ca 1-1000 mGy), and killed after nine months at which time the thyroid and blood samples were collected. Gene expression microarray analysis (thyroid samples) and LC-MS/MS analysis (thyroid and plasma samples) were performed to assess differential gene and protein expression profiles in treated and corresponding untreated control samples. Bioinformatics analyses were performed using the DAVID functional annotation tool and Ingenuity Pathway Analysis (IPA). The gene expression microarray data and LC-MS/MS data were validated using qRT-PCR and ELISA, respectively. RESULTS: Nine 131I exposure-related candidate biomarkers (transcripts: Afp and RT1-Bb, and proteins: ARF3, DLD, IKBKB, NONO, RAB6A, RPN2, and SLC25A5) were identified in thyroid tissue. Two dose-related protein candidate biomarkers were identified in thyroid (APRT and LDHA) and two in plasma (DSG4 and TGM3). Candidate biomarkers for thyroid function included the ACADL and SORBS2 (all activities), TPO and TG proteins (low activities). 131I exposure was shown to have a profound effect on metabolism, immune system, apoptosis and cell death. Furthermore, several signalling pathways essential for normal cellular function (actin cytoskeleton signalling, HGF signalling, NRF2-mediated oxidative stress, integrin signalling, calcium signalling) were also significantly regulated. CONCLUSION: Exposure-related and dose-related effects on gene and protein expression generated few expression patterns useful as biomarkers for thyroid function and cancer.

Biodistribution of 131I in mice is influenced by circadian variations.

Effects of radiation and biodistribution of radionuclides are often studied in animal models. Circadian rhythm affects many biological functions and may influence the biokinetics of radionuclides and observed responses. The aim of this study was to investigate if the time during the day of 131I injection affects the biodistribution and absorbed dose to tissues in mice. Biodistribution studies were conducted on male C57BL/6 N mice for three diurnal time-series: the animals were i.v. injected with 160 kBq 131I at 8 am, 12 pm or 4 pm. The activity concentration in organs and tissues was measured at 1 h to 7 days after administration and absorbed dose at day 7 was determined. Comparison between the three time-series showed statistically significant differences in activity concentration in all investigated tissues and organs. Administration performed at 12 pm resulted in general in higher absorbed dose to the organs than injection performed at 8 am and 4 pm. Time of day of administration affects the biodistribution of 131I in mice and consequently the absorbed dose to individual organs. These findings advocate that subsequent biodistribution studies and dosimetry calculations should consider time-point of administration as a variable that could influence the results.

Genomic profiling of the transcription factor Zfp148 and its impact on the p53 pathway.

Recent data suggest that the transcription factor Zfp148 represses activation of the tumor suppressor p53 in mice and that therapeutic targeting of the human orthologue ZNF148 could activate the p53 pathway without causing detrimental side effects. We have previously shown that Zfp148 deficiency promotes p53-dependent proliferation arrest of mouse embryonic fibroblasts (MEFs), but the underlying mechanism is not clear. Here, we showed that Zfp148 deficiency downregulated cell cycle genes in MEFs in a p53-dependent manner. Proliferation arrest of Zfp148-deficient cells required increased expression of ARF, a potent activator of the p53 pathway. Chromatin immunoprecipitation showed that Zfp148 bound to the ARF promoter, suggesting that Zfp148 represses ARF transcription. However, Zfp148 preferentially bound to promoters of other transcription factors, indicating that deletion of Zfp148 may have pleiotropic effects that activate ARF and p53 indirectly. In line with this, we found no evidence of genetic interaction between TP53 and ZNF148 in CRISPR and siRNA screen data from hundreds of human cancer cell lines. We conclude that Zfp148 deficiency, by increasing ARF transcription, downregulates cell cycle genes and cell proliferation in a p53-dependent manner. However, the lack of genetic interaction between ZNF148 and TP53 in human cancer cells suggests that therapeutic targeting of ZNF148 may not increase p53 activity in humans.

The IRI-DICE hypothesis: ionizing radiation-induced DSBs may have a functional role for non-deterministic responses at low doses.

Low-dose ionizing radiation (IR) responses remain an unresolved issue in radiation biology and risk assessment. Accurate knowledge of low-dose responses is important for estimation of normal tissue risk in cancer radiotherapy or health risks from occupational or hazard exposure. Cellular responses to low-dose IR appear diverse and stochastic in nature and to date no model has been proposed to explain the underlying mechanisms. Here, we propose a hypothesis on IR-induced double-strand break (DSB)-induced cis effects (IRI-DICE) and introduce DNA sequence functionality as a submicron-scale target site with functional outcome on gene expression: DSB induction in a certain genetic target site such as promotor, regulatory element, or gene core would lead to changes in transcript expression, which may range from suppression to overexpression depending on which functional element was damaged. The DNA damage recognition and repair machinery depicts threshold behavior requiring a certain number of DSBs for induction. Stochastically distributed persistent disruption of gene expression may explain-in part-the diverse nature of low-dose responses until the repair machinery is initiated at increased absorbed dose. Radiation quality and complexity of DSB lesions are also discussed. Currently, there are no technologies available to irradiate specific genetic sites to test the IRI-DICE hypothesis directly. However, supportive evidence may be achieved by developing a computational model that combines radiation transport codes with a genomic DNA model that includes sequence functionality and transcription to simulate expression changes in an irradiated cell population. To the best of our knowledge, IRI-DICE is the first hypothesis that includes sequence functionality of different genetic elements in the radiation response and provides a model for the diversity of radiation responses in the (very) low dose regimen.

Increased therapeutic effect on medullary thyroid cancer using a combination of radiation and tyrosine kinase inhibitors.

Since patients with medullary thyroid cancer (MTC) often have metastatic disease at the time of diagnosis, the development of efficient systemic treatment options for MTC is important. Vandetanib and cabozantinib are two tyrosine kinase inhibitors (TKIs) that were recently approved by FDA and EMA for systemic treatment of metastatic MTC. Additionally, since MTC is of a neuroendocrine tumour type, treatment with radiolabelled somatostatin analogues (e.g. 177Lu-octreotate) is a valid option for patients with MTC. The aim of this study was to investigate the potentially increased therapeutic effect of combining radiation therapy with these TKIs for treatment of MTC in a mouse model. Nude mice carrying patient-derived MTC tumours (GOT2) were treated with external beam radiotherapy (EBRT) and/or one of the two TKIs vandetanib or cabozantinib. The tumour volume was determined and compared with that of mock-treated controls. The treatment doses were chosen to give a moderate effect as monotherapy to be able to detect any increased therapeutic effect from the combination therapy. At the end of follow-up, tumours were processed for immunohistochemical (IHC) analyses. The animals in the combination therapy groups showed the largest reduction in tumour volume and the longest time to tumour progression. Two weeks after start of treatment, the tumour volume for these mice was reduced by about 70-75% compared with controls. Furthermore, also EBRT and TKI monotherapy resulted in a clear anti-tumour effect with a reduced tumour growth compared with controls. The results show that an increased therapeutic effect could be achieved when irradiation is combined with TKIs for treatment of MTC. Future studies should evaluate the potential of using 177Lu-octreotate therapy in combination with TKIs in patients.