UBL3 overexpression enhances EV-mediated Achilles protein secretion in conditioned media of MDA-MB-231 cells.

Cancer cells communicate within the tumor microenvironment (TME) through extracellular vesicles (EVs), which act as crucial messengers in intercellular communication, transporting biomolecules to facilitate cancer progression. Ubiquitin-like 3 (UBL3) facilitates protein sorting into small EVs as a post-translational modifier. However, the effect of UBL3 overexpression in EV-mediated protein secretion has not been investigated yet. This study aimed to investigate the effect of UBL3 overexpression in enhancing EV-mediated Achilles protein secretion in MDA-MB-231 (MM) cells by a dual-reporter system integrating Akaluc and Achilles tagged with Ubiquitin where self-cleaving P2A linker connects Akaluc and Achilles. MM cells stably expressing Ubiquitin-Akaluc-P2A-Achilles (Ubi-Aka/Achi) were generated. In our study, both the bioluminescence of Ubiquitin-Akaluc (Ubi-Aka) and the fluorescence of Achilles secretion were observed. The intensity of Ubi-Aka was thirty times lower, while the Achilles was four times lower than the intensity of corresponding cells. The ratio of Ubi-Aka and Achilles in conditioned media (CM) was 7.5. They were also detected within EVs using an EV uptake luciferase assay and fluorescence imaging. To investigate the effect of the UBL3 overexpression in CM, Ubi-Aka/Achi was transiently transfected into MM-UBL3-KO, MM, and MM-Flag-UBL3 cells. We found that the relative fluorescence expression of Achilles in CM of MM-UBL3-KO, MM, and MM-Flag-UBL3 cells was 30 %, 28 %, and 45 %, respectively. These findings demonstrated that UBL3 overexpression enhances EV-mediated Achilles protein secretion in CM of MM cells. Targeting UBL3 could lead to novel therapies for cancer metastasis by reducing the secretion of pro-metastatic proteins, thereby inhibiting disease progression.

Status of incremental costs of first-line treatment recommended in Japanese clinical guidelines for metastatic breast cancer patients.

BACKGROUND: The increasing incidence and prevalence of breast cancer alongside diagnostic and treatment technology advances have produced a debate about the financial burden cancer places on the healthcare system and concerns about access. METHODS: This study was conducted at 51 hospitals belonging to the Breast Cancer Study Group of the Japan Clinical Oncology Group using a web-based survey. The survey period conducted from July 2021 to June 2022. The study population included patients with metastatic breast cancer who received the related treatment as their first-line therapy. The proportion of patients who selected that regimen as their first-line treatment was tabulated. The total cost increase for each current standard therapy in comparison to conventional treatments was calculated. RESULTS: A total of 702 patients (pts) were surveyed. Of those enrolled, 342 (48.7%) received high-cost treatment [estimated monthly drug costs exceeding ~500 000 Japanese Yen (JPY)]. Of these, 16 pts (4.7%) were receiving very high-cost treatment, amounting to more than 1 000 000 JPY per month. Fifty three (15.5%) of the patients who received high-cost treatment were 75 years of age or older. Of these, 1 pt (0.3%) were receiving very high-cost treatment. Analyses of incremental costs by current drugs showed that abemaciclib was costly with total additional cost of 6 365 670 JPY per patient. The total additional cost of the regimen per patient that included palbociclib was the second highest at 4011248 JPY, followed by atezolizumab at 3209033 JPY. CONCLUSIONS: The findings indicate that evaluating the financial implications of high-cost treatments requires considering not only drug prices but also analysis of total cost increase.

Ubiquitin-like 3 as a new protein-sorting factor for small extracellular vesicles.

Ubiquitin-like 3 (UBL3) is a well-conserved ubiquitin-like protein (UBL) in eukaryotes and regulates the ubiquitin cascade, but the significant roles of UBL3 in cellular processes remained unknown. Recently, UBL3 was elucidated to be a post-translational modification factor that promotes protein sorting to small extracellular vesicles (sEVs). Proteins sorted into sEVs have been studied as etiologies of sEV-related diseases. Also, there have been attempts to construct drug delivery systems (DDSs) by loading proteins into sEVs. In this review, we introduce the new concept that UBL3 has a critical role in the protein-sorting system and compare structure conservation between UBL3 and other UBLs from an evolutionary perspective. We conclude with future perspectives for the utility of UBL3 in sEV-related diseases and DDS.Key words: UBL3, small extracellular vesicles, protein sorting, ubiquitin-like protein, post-translational modification.

DNA methylation alterations of AXIN2 in serrated adenomas and colon carcinomas with microsatellite instability.

BACKGROUND: Recent work led to recognize sessile serrated adenomas (SSA) as precursor to many of the sporadic colorectal cancers with microsatellite instability (MSI). However, comprehensive analyses of DNA methylation in SSA and MSI cancer have not been conducted. METHODS: With an array-based methylation sensitive amplified fragment length polymorphism (MS-AFLP) method we analyzed 8 tubular (TA) and 19 serrated (SSA) adenomas, and 14 carcinomas with (MSI) and 12 without (MSS) microsatellite instability. MS-AFLP array can survey relative differences in methylation between normal and tumor tissues of 9,654 DNA fragments containing all NotI sequences in the human genome. RESULTS: Unsupervised clustering analysis of the genome-wide hypermethylation alterations revealed no major differences between or within these groups of benign and malignant tumors regardless of their location in intergenic, intragenic, promoter, or 3' end regions. Hypomethylation was less frequent in SSAs compared with MSI or MSS carcinomas. Analysis of variance of DNA methylation between these four subgroups identified 56 probes differentially altered. The hierarchical tree of this subset of probes revealed two distinct clusters: Group 1, mostly composed by TAs and MSS cancers with KRAS mutations; and Group 2 with BRAF mutations, which consisted of cancers with MSI and MLH1 methylation (Group 2A), and SSAs without MLH1 methylation (Group 2B). AXIN2, which cooperates with APC and ß-catenin in Wnt signaling, had more methylation alterations in Group 2, and its expression levels negatively correlated with methylation determined by bisulfite sequencing. Within group 2B, low and high AXIN2 expression levels correlated significantly with differences in size (P = 0.01) location (P = 0.05) and crypt architecture (P = 0.01). CONCLUSIONS: Somatic methylation alterations of AXIN2, associated with changes in its expression, stratify SSAs according to some clinico-pathological differences. We conclude that hypermethylation of MLH1, when occurs in an adenoma cell with BRAF oncogenic mutational activation, drives the pathway for MSI cancer by providing the cells with a mutator phenotype. AXIN2 inactivation may contribute to this tumorigenic pathway either by mutator phenotype driven frameshift mutations or by epigenetic deregulation contemporary with the unfolding of the mutator phenotype.

Neurofibroma with adenosis in the mammary gland: a case report.

BACKGROUND: Neurofibroma of the breast is extremely rare, with only a few reported cases. Here, we report a case of solitary neurofibroma of the breast in a 95-year-old woman. CASE PRESENTATION: A 95-year-old woman presented with a palpable mass in the left breast. Mammography revealed a well-defined mass. A 1.6-cm round mass was found in the lower outer quadrant of the left breast on ultrasonography. The internal echo of the tumor was a mixture of relatively uniform hypoechoic areas with posterior enhancement and heterogeneous hyperechoic areas. She underwent a core needle biopsy. The pathological findings revealed a spindle cell lesion with no malignant findings. At 2 months follow-up, repeat breast ultrasonography showed that the mass had enlarged to be 2.7 cm in size. A repeat core needle biopsy, however, revealed no particularly new information. Because the tumor was growing and a definite diagnosis was not made, lumpectomy was performed. We found bland-spindled cells with shredded-carrot collagen bundles. Immunohistochemical antibody markers (S100, SOX10, and CD34) were positive for the spindle cells. Some of the tumors maintained the bilayer nature of luminal cells and myoepithelial cells, which might be the reason for internal heterogeneity on ultrasound. A histological diagnosis of neurofibroma with adenosis was made. At 6 months follow-up, no recurrent lesions were found. CONCLUSIONS: Ultrasound and pathological images revealed an extremely rare case of neurofibroma combined with adenosis. Tumor resection was performed because it was difficult to make a definitive diagnosis using needle biopsy. Even when a benign tumor is suspected, short-term follow-up is necessary, and if an enlargement is observed, early tumor resection is recommended.

Lipidomics-based tissue heterogeneity in specimens of luminal breast cancer revealed by clustering analysis of mass spectrometry imaging: A preliminary study.

Cancer tissues reflect a greater number of pathological characteristics of cancer compared to cancer cells, so the evaluation of cancer tissues can be effective in determining cancer treatment strategies. Mass spectrometry imaging (MSI) can evaluate cancer tissues and even identify molecules while preserving spatial information. Cluster analysis of cancer tissues' MSI data is currently used to evaluate the phenotype heterogeneity of the tissues. Interestingly, it has been reported that phenotype heterogeneity does not always coincide with genotype heterogeneity in HER2-positive breast cancer. We thus investigated the phenotype heterogeneity of luminal breast cancer, which is generally known to have few gene mutations. As a result, we identified phenotype heterogeneity based on lipidomics in luminal breast cancer tissues. Clusters were composed of phosphatidylcholine (PC), triglycerides (TG), phosphatidylethanolamine, sphingomyelin, and ceramide. It was found that mainly the proportion of PC and TG correlated with the proportion of cancer and stroma on HE images. Furthermore, the number of carbons in these lipid class varied from cluster to cluster. This was consistent with the fact that enzymes that synthesize long-chain fatty acids are increased through cancer metabolism. It was then thought that clusters containing PCs with high carbon counts might reflect high malignancy. These results indicate that lipidomics-based phenotype heterogeneity could potentially be used to classify cancer for which genetic analysis alone is insufficient for classification.

The Japanese Breast Cancer Society Clinical Practice Guidelines for systemic treatment of breast cancer, 2022 edition.

The Japanese Breast Cancer Society (JBCS) Clinical Practice Guidelines for systemic treatment of breast cancer were updated to the 2022 edition through a process started in 2018. The updated guidelines consist of 12 background questions (BQs), 33 clinical questions (CQs), and 20 future research questions (FRQs). Multiple outcomes including efficacy and safety were selected in each CQ, and then quantitative and qualitative systematic reviews were conducted to determine the strength of evidence and strength of recommendation, which was finally determined through a voting process among designated committee members. Here, we describe eight selected CQs as important updates from the previous guidelines, including novel practice-changing updates, and recommendations based on evidence that has emerged specifically from Japanese clinical trials.

A difficult-to-diagnose fibromatosis-like metaplastic carcinoma of the breast: a case report.

BACKGROUND: Fibromatosis-like metaplastic carcinoma (FLMCa), classified as a metaplastic carcinoma of the breast, is a very rare type of metaplastic carcinoma. We report a case of FLMCa that was difficult to diagnose. CASE PRESENTATION: The patient was a 56-year-old postmenopausal woman who presented with a left-sided breast mass. A 1.3-cm irregular mass was found in the lower outer quadrant of the left breast on breast ultrasonography. She underwent core needle biopsy and vacuum-assisted biopsy, but the pathological findings only revealed inflammatory cell infiltration and a high level of fibrosis, with no malignant findings. At 3 months follow-up, she underwent a repeat breast ultrasonography, which revealed an increase in the size of the mass to 1.8 cm, and a repeat core needle biopsy, which showed a few spindle cells and squamous cells positive for cytokeratin (CK)5/6 and AE1/AE3, leading to the suspicion of FLMCa. Since the amount of tissue was insufficient to establish a definitive diagnosis, she underwent a lumpectomy. We found low-grade and slightly atypical spindle cells and partly atypical spindle cell carcinoma and squamous cell carcinoma. CK5/6 and α-SMA were positive, thus confirming FLMCa. Because the margins on the edge of the nipple side and anterior side were "ink on tumor", she underwent a mastectomy and sentinel lymph node biopsy. After the surgery, she received adjuvant chemotherapy. At 3 years and 8 months of follow-up, no recurrent or metastatic lesions were identified in her body. CONCLUSIONS: FLMCa should be considered in the differential diagnosis when collagenous fibers are proliferating and malignancy is clinically suspected. Immunohistochemical analysis may be helpful in confirming this diagnosis.

Dose-dense adjuvant chemotherapy: a systematic review and meta-analysis of the Japanese Breast Cancer Society Clinical Practice Guideline, 2018 edition.

Increasing the dose intensity of cytotoxic therapy by shortening the intervals between cycles may enhance efficacy. Dose-dense chemotherapy, which is adopted as adjuvant chemotherapy of high-risk breast cancer, is addressed in the Japanese Breast Cancer Society Clinical Practice Guideline for breast cancer, 2018 edition (in Japanese). To evaluate the benefits and safety of dose-dense adjuvant chemotherapy described in the guideline, we performed a systematic review and meta-analysis of data of randomized trials using the same drugs, doses, and numbers of cycles. The PubMed, Cochrane Library, and Ichushi-Web databases were searched for relevant publications reporting randomized trials published until November 2016. Overall survival (OS), disease-free survival (DFS), and toxicity were assessed. Three trials comprising 5190 patients were included. Compared with conventional chemotherapy, dose-dense chemotherapy lengthened OS (RR = 0.76; 95% CI = 0.64-0.90) and DFS (RR = 0.83; 95% CI = 0.75-0.92) and increased the risk of anemia (RR = 4.56; 95% CI = 2.01-10.34). We conclude that dose-dense chemotherapy can be highly recommended as adjuvant chemotherapy for patients with breast cancer with a high risk of recurrence risk and sufficient bone marrow function.

Preoperative concurrent endocrine therapy with chemotherapy in luminal B-like breast cancer.

PURPOSE: Luminal B-like breast cancer is sensitive to both chemotherapy and endocrine therapy. We aimed to assess the safety and efficacy of concomitant chemotherapy and endocrine therapy compared with chemotherapy alone in the preoperative setting in luminal B-like breast cancer. METHODS: This two-arm randomized clinical trial enrolled patients with luminal B-like human epithelial growth factor 2-negative breast cancer, who were randomly assigned at a 1:1 ratio to receive preoperative chemotherapy alone or preoperative endocrine therapy concurrent with chemotherapy for 24 weeks before surgery. The primary endpoint was the pathological complete response (pCR) rate. The secondary endpoints included the clinical response rate, toxicity, and health-related quality of life (HRQOL). RESULTS: Overall, 70 patients were randomly assigned to the chemotherapy and chemo-endocrine therapy groups. The pCR rates were 9.7% and 3.0% (P = 0.319), and the clinical complete response rates were 5.9% and 5.6% (P = 0.745) in the chemotherapy and chemo-endocrine therapy groups, respectively. There were no clear differences in treatment-related adverse events or HRQOL between the two groups. CONCLUSIONS: In patients with luminal B-like breast cancer, the pCR, clinical response rate, toxicity, and HRQOL with the concomitant administration of endocrine therapy and chemotherapy were not superior to chemotherapy alone in the preoperative setting.