Intratumor transforming growth factor-ß signaling with extracellular matrix-related gene regulation marks chemotherapy-resistant gastric cancer.

Drug-tolerant persister (DTP) cells remain following chemotherapy and can cause cancer relapse. However, it is unclear when acquired resistance to chemotherapy emerges. Here, we compared the gene expression profiles of gastric cancer patient-derived cells (GC PDCs) and their respective xenograft tumors with different sensitivities to 5-fluorouracil (5-FU) by using immunodeficient female BALB/c-nu mice. RNA sequencing analysis of 5-FU-treated PDCs demonstrated that DNA replication/cell cycle-related genes were transiently induced in the earlier phase of DTP cell emergence, while extracellular matrix (ECM)-related genes were sustainably upregulated during long-term cell survival in 5-FU-resistant residual tumors. NicheNet analysis, which uncovers cell-cell signal interactions, indicated the transforming growth factor-ß (TGF-ß) pathway as the upstream regulator in response to 5-FU treatment. This induced ECM-related gene expression in the 5-FU-resistant tumor model. In the 5-FU-resistant residual tumors, there was a marked upregulation of cancer cell-derived TGF-ß1 expression and increased phosphorylation of SMAD3, a downstream regulator of the TGF-ß receptor. By contrast, these responses were not observed in a 5-FU-sensitive tumor model. We further found that TGF-ß-related upregulation of ECM genes was preferentially observed in non-responders to chemotherapy with 5-FU and/or oxaliplatin among 22 patient-derived xenograft tumors. These observations suggest that chemotherapy-induced activation of the TGF-ß1/SMAD3/ECM-related gene axis is a potential biomarker for the emergence of drug resistance in GCs.

Pharmacologic Targeting of Histone H3K27 Acetylation/BRD4-dependent Induction of ALDH1A3 for Early-phase Drug Tolerance of Gastric Cancer.

Anticancer drug-tolerant persister (DTP) cells at an early phase of chemotherapy reshape refractory tumors. Aldehyde dehydrogenase 1 family member A3 (ALDH1A3) is commonly upregulated by various anticancer drugs in gastric cancer patient-derived cells (PDC) and promotes tumor growth. However, the mechanism underlying the generation of ALDH1A3-positive DTP cells remains elusive. Here, we investigated the mechanism of ALDH1A3 expression and a combination therapy targeting gastric cancer DTP cells. We found that gastric cancer tissues treated with neoadjuvant chemotherapy showed high ALDH1A3 expression. Chromatin immunoprecipitation (ChIP)-PCR and ChIP sequencing analyses revealed that histone H3 lysine 27 acetylation was enriched in the ALDH1A3 promoter in 5-fluorouracil (5-FU)-tolerant persister PDCs. By chemical library screening, we found that the bromodomain and extraterminal (BET) inhibitors OTX015/birabresib and I-BET-762/molibresib suppressed DTP-related ALDH1A3 expression and preferentially inhibited DTP cell growth. In DTP cells, BRD4, but not BRD2/3, was recruited to the ALDH1A3 promoter and BRD4 knockdown decreased drug-induced ALDH1A3 upregulation. Combination therapy with 5-FU and OTX015 significantly suppressed in vivo tumor growth. These observations suggest that BET inhibitors are efficient DTP cell-targeting agents for gastric cancer treatment. SIGNIFICANCE: Drug resistance hampers the cure of patients with cancer. To prevent stable drug resistance, DTP cancer cells are rational therapeutic targets that emerge during the early phase of chemotherapy. This study proposes that the epigenetic regulation by BET inhibitors may be a rational therapeutic strategy to eliminate DTP cells.

APC/PIK3CA mutations and ß-catenin status predict tankyrase inhibitor sensitivity of patient-derived colorectal cancer cells.

BACKGROUND: Aberrant WNT/ß-catenin signaling drives carcinogenesis. Tankyrases poly(ADP-ribosyl)ate and destabilize AXINs, ß-catenin repressors. Tankyrase inhibitors block WNT/ß-catenin signaling and colorectal cancer (CRC) growth. We previously reported that 'short' APC mutations, lacking all seven ß-catenin-binding 20-amino acid repeats (20-AARs), are potential predictive biomarkers for CRC cell sensitivity to tankyrase inhibitors. Meanwhile, 'Long' APC mutations, which possess more than one 20-AAR, do not predict inhibitor-resistant cells. Thus, additional biomarkers are needed to precisely predict the inhibitor sensitivity. METHODS: Using 47 CRC patient-derived cells (PDCs), we examined correlations between the sensitivity to tankyrase inhibitors (G007-LK and RK-582), driver mutations, and the expressions of signaling factors. NOD.CB17-Prkdcscid/J and BALB/c-nu/nu xenograft mice were treated with RK-582. RESULTS: Short APC mutant CRC cells exhibited high/intermediate sensitivities to tankyrase inhibitors in vitro and in vivo. Active ß-catenin levels correlated with inhibitor sensitivity in both short and long APC mutant PDCs. PIK3CA mutations, but not KRAS/BRAF mutations, were more frequent in inhibitor-resistant PDCs. Some wild-type APC PDCs showed inhibitor sensitivity in a ß-catenin-independent manner. CONCLUSIONS: APC/PIK3CA mutations and ß-catenin predict the sensitivity of APC-mutated CRC PDCs to tankyrase inhibitors. These observations may help inform the strategy of patient selection in future clinical trials of tankyrase inhibitors.

Locus Coeruleus-Noradrenergic Neurons Regulate Stress Coping During Subchronic Exposure to Social Threats: A Characteristic Feature in Postpartum Female Mice.

Stress-coping strategies have been implicated in depression. The control of stress coping may improve the symptom and higher prevalence of depression during the postpartum period in women. However, the neuronal mechanisms underlying stress coping remain to be fully elucidated in postpartum women. In this study, we examined how locus coeruleus-noradrenergic (LC-NA) neurons, which have been associated with both stress coping and depression, regulate changes in coping style induced by subchronic exposure to unfamiliar male mice as a social threat in postpartum female mice. In contrast to virgin females, dams exposed to unfamiliar males daily for four consecutive days showed reduced immobility duration in the forced swim test, indicating that exposure to unfamiliar males decreased passive stress coping in dams. Exposure to unfamiliar males also decreased sucrose palatability in the sucrose preference test and suppressed the crouching behavior in the maternal care test but did not affect anxiety-like behavior in the hole-board test in dams. In fiber photometry analyses, LC-NA neurons showed differential activity between dams and virgin females in response to unfamiliar males. Chemogenetic inhibition of LC-NA neurons during exposure to unfamiliar males prevented the social threat-induced decrease in immobility duration in the forced swim test in dams. Furthermore, inhibition or activation of LC-NA neurons exacerbated crouching behavior in dams. These results indicate that LC-NA neurons regulate the social threat-induced decrease in passive stress coping and relieve social threat-induced inhibition of maternal care in postpartum female mice.

Fatal hemoperitoneum due to rupture of mesenteric artery in remission state of microscopic polyangiitis, concomitant with severe hypertension and posterior reversible encephalopathy syndrome: an autopsy case report.

Microscopic polyangiitis (MPA) is a type of necrotizing vasculitis associated with high levels of myeloperoxidase-specific antineutrophil cytoplasmic antibody (MPO-ANCA). While generally associated with renal dysfunction, MPA can also cause intraabdominal hemorrhage in rare cases. A 66-year-old man was admitted to our hospital for renal dysfunction, numbness, and weight loss for 3 months. He had no significant medical history. Renal biopsy revealed crescentic glomerulonephritis with necrotizing vasculitis, which was associated with a high serum titer of MPO-ANCA, leading to a diagnosis of MPA. Remission-induction treatment with glucocorticoids and rituximab was initiated, which improved the patient's general condition and renal failure. His blood pressure was elevated and was controlled by amlodipine treatment. Two months after discharge, he visited the emergency department because of chest pain. A diagnosis of acute cardiovascular syndrome was suggested; however, his cardiac artery was not stenotic. The patient's blood pressure was high despite antihypertensive therapy, and he developed posterior reversible encephalopathy syndrome (PRES). Despite intensive treatment, the patient died 3 days later. An autopsy revealed that the cause of death was hypovolemic shock due to massive intra-abdominal hemorrhage from the ruptured mesenteric artery involved in vasculitis. In cases of MPA with sudden-onset chest or abdominal pain, a ruptured intra-abdominal artery should be considered. Secondary hypertension associated with vasculitis should be carefully managed to prevent hemorrhagic complications and PRES.

Investigation of the factors that induce maternal aggression towards juveniles among several mouse strains.

Maternal care and aggression are representative of maternal behavior among lactating female mice. Even neonates and juveniles, who are not biological offspring, can induce maternal care and aggression in dams. Here, we investigated the factors that induce maternal aggression through exposure to juvenile mice. We first addressed the role of intruder age on the induction of maternal aggression in dams. BALB/c dams displayed attacking behavior towards 14-day-old C57BL/6J male intruders. Consumption of food pellets during the weaning period was unlikely to affect the induction of attacking behavior, as the intruders reared by breastfeeding, without food pellets, induced intensive attacking behavior in dams. Next, we compared the intruder-mediated induction of attacking behavior through different mouse strains. Specifically, BALB/c intruders induced a lower level of attacking behavior in BALB/c or ICR dams, compared to the other strains tested. However, BALB/c intruders induced intense attacking behavior in C57BL/6N dams, indicating that the occurrence of attacking behavior is dependent on the strains of dams as well as intruders. A cross-fostering experiment highlighted that the rearing by an original mother was required for C57BL/6J juveniles to induce attacking behavior. In contrast, BALB/c intruders may emit an inhibitory factor that limits attacking behavior. We finally explored which parts of the body emit these aggression-inducible signals. Removal of body hair around the proximal tail of the intruders significantly decreased the attacking behavior of dams, demonstrating that chemical cues, namely pheromones, attached to the body hair around the proximal tail may be essential for inducing attacking behavior in dams.

Oxytocin in the dorsal raphe nucleus antagonizes the inhibition of maternal care induced by food deprivation.

Lactation is indispensable for the pup's survival, but is considered a survival burden in dams under negative energy conditions. In the present study, we tested our hypothesis that oxytocin may facilitate energy investment to pups through behavioral control as well as milk ejection. Maternal care was observed in dams at 3 h but not 8 h after food deprivation. We investigated whether oxytocin in the dorsal raphe nucleus (DRN), which is involved in energy state-dependent regulation of maternal care, regulates maternal care. For this purpose, 2-pmol L368899, an oxytocin receptor antagonist, was injected into the DRN; after treatment, maternal care was inhibited in the dams with 3-h fasting, but not in the fed dams. In contrast, recovery of maternal care was observed in the dams with 8-h fasting who underwent 100-pmol oxytocin injection at the DRN. These results indicate that oxytocin in the DRN is required for displaying maternal behavior under fasting conditions, but not under fed conditions. Next, we investigated the site of oxytocin release. Presentation of pups decreased the oxytocin immunoreactivity at the paraventricular nucleus (PVN) of the hypothalamus in the 3-h-fasted dams, but not in the fed or 8-h-fasted dams. No change of the serum oxytocin level was observed. Few oxytocin-positive neurons projecting from the PVN to the DRN were detected through labeling with the retrograde tracer fluorogold. Oxytocin secreted at the PVN, which reaches the DRN, but not released as a hormone or neurotransmitter may mediate maternal care under food-restricted conditions.

Nuclear Accumulation of HSP70 in Mouse Skeletal Muscles in Response to Heat Stress, Aging, and Unloading With or Without Reloading.

The purpose of this study was to investigate the nuclear accumulation of heat shock protein 70 (HSP70), a molecular chaperonin in mouse skeletal muscle in response to aging, heat stress, and hindlimb unloading with or without reloading. Profiles of HSP70-specific nuclear transporter Hikeshi in skeletal muscles were also evaluated. Heat stress-associated nuclear accumulation of HSP70 was observed in slow soleus (SOL) and fast plantaris (PLA) muscles of young (10-week-old) mice. Mean nuclear expression level of HSP70 in slow medial gastrocnemius (MGAS) and PLA muscles of aged (100-week-old) mice increased ~4.8 and ~1.7 times, compared to that of young (10-week-old) mice. Reloading following 2-week hindlimb unloading caused accumulation of HSP70 in myonuclei in MGAS and PLA of young mice ( p < 0.05). However, reloading-associated nuclear accumulation of HSP70 was not observed in both types of muscles of aged mice. On the other hand, 2-week hindlimb unloading had no impact on the nuclear accumulation of HSP70 in both muscles of young and aged mice. Nuclear expression level of Hikeshi in both MGAS and PLA in mice was suppressed by aging. No significant changes in the nuclear Hikeshi in both muscles were induced by unloading with or without reloading. Results of this study indicate that the nuclear accumulation of HSP70 might show a protective response against cellular stresses in skeletal muscle and that the protective response may be suppressed by aging. Protective response to aging might depend on muscle fiber types.

Microcurrent electrical neuromuscular stimulation facilitates regeneration of injured skeletal muscle in mice.

Conservative therapies, mainly resting care for the damaged muscle, are generally used as a treatment for skeletal muscle injuries (such as muscle fragmentation). Several past studies reported that microcurrent electrical neuromuscular stimulation (MENS) facilitates a repair of injured soft tissues and shortens the recovery period. However, the effects of MENS on the regeneration in injured skeletal muscle are still unclear. The purpose of this study was to investigate the effect of MENS on the regenerative process of injured skeletal muscle and to elucidate whether satellite cells in injured skeletal muscle are activated by MENS by using animal models. Male C57BL/6J mice, aged 7 weeks old, were used (n = 30). Mice were randomly divided into two groups: (1) cardiotoxin (CTX)-injected (CX, n = 15) and (2) CTX-injected with MENS treatment (MX, n=15) groups. CTX was injected into tibialis anterior muscle (TA) of mice in CX and MX groups to initiate the necrosis-regeneration cycle of the muscle. TA was dissected 1, 2, and 3 weeks after the injection. Muscle weight, muscle protein content, the mean cross-sectional areas of muscle fibers, the relative percentage of fibers having central nuclei, and the number of muscle satellite cells were evaluated. MENS facilitated the recovery of the muscle dry weight and protein content relative to body weight, and the mean cross-sectional areas of muscle fibers in CTX-induced injured TA muscle. The number of Pax7-positive muscle satellite cells was increased by MENS during the regenerating period. Decrease in the percentages of fibers with central nuclei after CTX-injection was facilitated by MENS. MENS may facilitate the regeneration of injured skeletal muscles by activating the regenerative potential of skeletal muscles. Key pointsMicrocurrent electrical neuromuscular stimulation (MENS) facilitated the recovery of the relative muscle dry weight, the relative muscle protein content, and the mean cross-sectional areas of muscle fibers of injured TA muscle in mice.The number of satellite cells was increased by MENS during the regenerating phase of injured skeletal muscle.Decrease in the percentages of fibers with central nuclei was facilitated by MENS.MENS may facilitate the regeneration of injured skeletal muscles.

Microcurrent electrical nerve stimulation facilitates regrowth of mouse soleus muscle.

Microcurrent electrical nerve stimulation (MENS) has been used to facilitate recovery from skeletal muscle injury. However, the effects of MENS on unloading-associated atrophied skeletal muscle remain unclear. Effects of MENS on the regrowing process of unloading-associated atrophied skeletal muscle were investigated. Male C57BL/6J mice (10-week old) were randomly assigned to untreated normal recovery (C) and MENS-treated (M) groups. Mice of both groups are subjected to continuous hindlimb suspension (HS) for 2 weeks followed by 7 days of ambulation recovery. Mice in M group were treated with MENS for 60 min 1, 3, and 5 days following HS, respectively, under anesthesia. The intensity, the frequency, and the pulse width of MENS were set at 10 µA, 0.3 Hz, and 250 msec, respectively. Soleus muscles were dissected before and immediately after, 1, 3 and 7 days after HS. Soleus muscle wet weight and protein content were decreased by HS. The regrowth of atrophied soleus muscle in M group was faster than that in C group. Decrease in the reloading-induced necrosis of atrophied soleus was facilitated by MENS. Significant increases in phosphorylated levels of p70 S6 kinase and protein kinase B (Akt) in M group were observed, compared with C group. These observations are consistent with that MENS facilitated regrowth of atrophied soleus muscle. MENS may be a potential extracellular stimulus to activate the intracellular signals involved in protein synthesis.

Prognostic values of initial responses to low-dose (131)I-MIBG therapy in patients with malignant pheochromocytoma and paraganglioma.

PURPOSE: We retrospectively examined whether or not initial responses of first low-dose (131)I-meta-iodo-benzyl-guanidine radiotherapy ((131)I-MIBG therapy) in patients with malignant pheochromocytoma and paraganglioma had prognostic values. MATERIALS AND METHODS: This study included 26 patients with malignant pheochromocytoma (n = 18) and paraganglioma (n = 8) who underwent the first (131)I-MIBG therapy between October 2001 and September 2007. Based on the initial subjective, hormonal, scintigraphic, and objective responses to (131)I-MIBG therapy, the responses were divided into progression disease (PD) and non-PD. We examined the following factors for prognostic significance: sex, age, disease, initial diagnosis (benign or malignant pheochromocytoma), hypertension, diabetes mellitus, palpitations, symptoms related to bone metastases, and number of low-dose (131)I-MIBG therapy. Univariate Cox proportional regression analysis was used to identify prognostic factors for overall survival. Overall survival was analyzed by Kaplan-Meier method and the curves were compared using the log-rank test. RESULTS: The median survival time was 56 months. In the follow-up period, 16 patients died from exacerbation of their diseases. Univariate analysis showed that the hormonal PD [hazard ratio (HR) 3.20, P = 0.034, confidence interval (CI) 1.09-9.93], objective PD (HR 11.89, P = 0.0068, CI 2.14-65.85), single-time (131)I-MIBG therapy (HR 3.22, P = 0.020, CI 1.21-8.79), hypertension (HR 2.93, P = 0.044, CI 1.02-10.50), and symptoms related to bone metastases (HR 3.54, P = 0.023, CI 1.18-13.04) were bad prognostic factors for overall survival. Kaplan-Meier analysis demonstrated that the hormonal non-PD (P = 0.026), objective non-PD (P = 0.0002), multiple-time (131)I-MIBG therapy (P = 0.013), and no symptom related to bone metastases (P = 0.024) were significantly associated with good prognosis. Overall survival rate was 70 and 50 % at 5 years from the initial diagnosis and from the first (131)I-MIBG therapy, respectively. CONCLUSION: The hormonal and objective responses to the first low-dose (131)I-MIBG therapy as well as complication of hypertension and symptoms related to bone metastases may be prognostic factors in patients with malignant pheochromocytoma and paraganglioma.

Thyroid hormone replacement one day before (131)I therapy in patients with well-differentiated thyroid cancer.

OBJECTIVE: The current study aimed to determine the efficacy of radioiodine-131 ((131)I) ablation therapy with thyroid hormone replacement one day before (131)I administration in patients with well-differentiated thyroid cancer (DTC). METHODS: This retrospective study included 29 patients who underwent (131)I therapies twice for DTC during 6-12 months. Since all the patients obviously had residual lesions by their serum thyroglobulin levels or their scintigrams at the first therapies, they underwent the second (131)I therapies without diagnostic scintigraphy after the first therapies. After confirming the sufficient elevation of TSH concentration, thyroid hormone replacement was resumed one day before (131)I administration (3.7-7.4GBq). The ablation rate of thyroid remnant at the first (131)I therapy was evaluated by comparing (131)I post-therapeutic images of the two treatments. RESULTS: Three patients were administrated thyroid hormone after (131)I therapy because of insufficient TSH concentration under thyroid hormone withdrawal. In the remaining 26 patients, 41 thyroid remnant accumulations were detected in all 26 patients at the first (131)I therapy. Based on the second (131)I post-therapeutic images, successful ablation was confirmed in 24 of 26 patients (92.3%) and 38 of 41 sites (92.7%), which was comparable with historically reported ablation rates. CONCLUSION: Thyroid hormone replacement one day before (131)I therapy could provide a sufficiently high ablation rate in patients with DTC.

I-131-Metaiodobenzylguanidine therapy with allogeneic cord blood stem cell transplantation for recurrent neuroblastoma.

Iodine-131-metaiodiobenzylguanidine (131I-MIBG) therapy combined with allogeneic cord blood stem cell transplantation (SCT) was used to treat a 4-year-old girl with recurrent neuroblastoma. The patient experienced relapse 2 years after receiving first-line therapies, which included chemotherapy, surgical resection, irradiation, and autologous peripheral SCT. Although 131I-MIBG treatment did not achieve complete remission, the size of the tumor was reduced after treatment. Based on our findings, we suggest that 131I-MIBG treatment with myeloablative allogeneic SCT should be considered as first-line therapy for high-risk neuroblastoma patients when possible.

Low-dose (123)I-metaiodobenzylguanidine diagnostic scan is inferior to (131)I-metaiodobenzylguanidine posttreatment scan in detection of malignant pheochromocytoma and paraganglioma.

OBJECTIVE: We assessed the lesion detectability of low-dose diagnostic (123)I-metaiodobenzylguanidine (MIBG) whole-body scans obtained at 6 and 24 h compared with posttreatment (131)I-MIBG whole-body scans in malignant pheochromocytoma and paraganglioma. METHODS: Scintigrams obtained in 15 patients with malignant pheochromocytoma and paraganglioma were retrospectively analyzed. Diagnostic scans were performed with 111 MBq of (123)I-MIBG. Therapeutic doses of (131)I-MIBG (5.55-7.40 GBq) were administrated and whole-body scans were obtained at 2-5 days after (131)I-MIBG administrations. We compared the number of lesions and the lesion-to-referent count ratios at 6 and 24 h of (123)I-MIBG and at 2-5 days of (131)I-MIBG. RESULTS: In comparison with the 6-h images of (123)I-MIBG, the 24-h images of (123)I-MIBG could detect more lesions in eight patients. Posttreatment (131)I-MIBG scans revealed new lesions in eight patients compared with the 24-h images of (123)I-MIBG. The lesion-to-referent count ratios at 6 and 24 h of (123)I-MIBG and at 3 days of (131)I-MIBG were increasing at later scanning time. There were significant differences in the lesion-to-referent count ratios between 6 and 24 h of (123)I-MIBG (P = 0.031), 6 h of (123)I-MIBG and 3 days of (131)I-MIBG (P = 0.020), and 24 h of (123)I-MIBG and 3 days of (131)I-MIBG (P = 0.018). CONCLUSION: Low-dose diagnostic (123)I-MIBG whole-body scan is inferior to posttreatment (131)I-MIBG whole-body scan in malignant pheochromocytoma and paraganglioma. Considering the scan timing of (123)I-MIBG, 6-h images might have no superiority compared with 24-h images.

Double-phase (131)I whole body scan and (131)I SPECT-CT images in patients with differentiated thyroid cancer: their effectiveness for accurate identification.

OBJECTIVE: This study aims to determine whether a (131)I double-phase whole body scan (WBS) and SPECT-CT images have added value over a single-phase WBS image in identifying benign and malignant lesions in patients with well-differentiated thyroid cancer (DTC) at their first radioactive iodine (RAI) treatment. METHODS: This study included 42 DTC patients who underwent their first radioablation. Post-therapeutic WBS images were acquired after 3 days (early phase) and 7 days (delayed phase). Following early-phase WBS, SPECT-CT images were obtained. The images were reviewed independently of the clinical data by 2 board-certified observers with a 6-point scoring system (benign to malignant -3 to +3). RESULTS: The double-phase WBS and SPECT-CT images showed 115 radioiodine-avid localizations (81 benign and 34 malignant accumulations). Confidence levels of benign accumulations were significantly higher with SPECT-CT (average score -2.40 ± 1.06) compared to those of the early-phase WBS (average score -1.39 ± 1.88) (p < 0.0001) and delayed-phase WBS images (average score -1.49 ± 1.19) (p < 0.0001). When the analysis was restricted to accumulations with a low confidence score in the early-phase WBS image, the confidence level of the delayed-phase WBS was higher compared to that of the early-phase WBS images (p = 0.0012). The confidence levels of malignant accumulations were significantly higher with SPECT-CT images (average score 2.37 ± 0.96) compared to the early-phase WBS (average score 1.44 ± 1.21) (p < 0.0001) and the delayed-phase WBS images (average score 1.50 ± 1.13) (p < 0.0001). CONCLUSION: Post-therapeutic SPECT-CT image was superior to the early-phase WBS image in enhancing the confidence level and accurately localizing the lesions. The delayed-phase WBS image contributed to the accurate diagnosis of benign lesions with a low confidence level in the early-phase WBS image.