A long-term survival case of histiocytic sarcoma by surgery alone in a Japanese elderly breast tumor patient.

INTRODUCTION: Histiocytic sarcoma (HS) is a rare hematologic malignancy. HS of the breast is extremely rare, and we present a case of an elderly patient with breast HS. CASE PRESENTATION: An 81-year-old woman with unremarkable past medical and family histories presented to our hospital with a palpable mass in her right upper breast. She had noticed a mass in her right breast 3 months before her first visit. Physical examination revealed a mass measuring approximately 30 mm in the right upper quadrant of the breast; there were no cervical or axillary lymphadenopathies. Mammography revealed a high-concentration mass with unclear margins in the upper and outer breast. Ultrasound and magnetic resonance imaging (MRI) revealed a 31 × 23-mm nodule with a relatively clear margin and necrotic sign on the T2-intensified image. A mastectomy was performed upon the patient's request, and the surgical specimen revealed a 35-mm hemorrhagic mass. The lesion was estrogen receptor-, progesterone receptor-, and HER2/neu-negative. The Ki-67 labeling index was approximately 30%. The immunohistochemical panel showed immune reactivity for the histiocytic markers CD68, CD163, and CD206 and was immune-negative for B lineage, T lineage, Langerhans cells, and keratins. The diagnosis of HS was based on the morphological and immunophenotypic characteristics of the mass. The patient received no systemic therapy and survived for 50 months without recurrence. CONCLUSIONS: Here, we report the case of an elderly patient with rare breast HS. Although the prognosis of HS seems poor, the breast HS was not as poor as expected, since it might have been discovered in the local region before it metastasized.

Changes in Triple-Negative Breast Cancer Molecular Subtypes in Patients Without Pathologic Complete Response After Neoadjuvant Systemic Chemotherapy.

PURPOSE: Lehmann et al have identified four molecular subtypes of triple-negative breast cancer (TNBC)-basal-like (BL) 1, BL2, mesenchymal (M), and luminal androgen receptor-and an immunomodulatory (IM) gene expression signature modifier. Our group previously showed that the response of TNBC to neoadjuvant systemic chemotherapy (NST) differs by molecular subtype, but whether NST affects the subtype was unknown. Here, we tested the hypothesis that in patients without pathologic complete response, TNBC subtypes can change after NST. Moreover, in cases with the changed subtype, we determined whether epithelial-to-mesenchymal transition (EMT) had occurred. MATERIALS AND METHODS: From the Pan-Pacific TNBC Consortium data set containing TNBC patient samples from four countries, we examined 64 formalin-fixed, paraffin-embedded pairs of matched pre- and post-NST tumor samples. The TNBC subtype was determined using the TNBCtype-IM assay. We analyzed a partial EMT gene expression scoring metric using mRNA data. RESULTS: Of the 64 matched pairs, 36 (56%) showed a change in the TNBC subtype after NST. The most frequent change was from BL1 to M subtypes (38%). No tumors changed from M to BL1. The IM signature was positive in 14 (22%) patients before NST and eight (12.5%) patients after NST. The EMT score increased after NST in 28 (78%) of the 36 patients with the changed subtype (v 39% of the 28 patients without change; P = .002254). CONCLUSION: We report, to our knowledge, for the first time that the TNBC molecular subtype and IM signature frequently change after NST. Our results also suggest that EMT is promoted by NST. Our findings may lead to innovative adjuvant therapy strategies in TNBC cases with residual tumor after NST.

Use of a Baculovirus-Mammalian Cell Expression-System for Expression of Drug-Metabolizing Enzymes: Optimization of Infection With a Focus on Cytochrome P450 3A4.

Heterologous expression systems are important for analyzing the effects of genetic factors including single nucleotide polymorphisms on the functions of drug-metabolizing enzymes. In this study, we focused on a baculovirus-mammalian cell (Bac-Mam) expression system as a safer and more efficient approach for this purpose. The baculovirus-insect cell expression system is widely utilized in large-scale protein expression. Baculovirus has been shown to also infect certain mammalian cells, although the virus only replicates in insect cells. With this knowledge, baculovirus is now being applied in a mammalian expression system called the Bac-Mam system wherein a gene-modified baculovirus is used whose promotor is replaced with one that can function in mammalian cells. We subcloned open-reading frames of cytochrome P450 3A4 (CYP3A4), UDP-glucuronosyltransferase (UGT) 1A1, and UGT2B7 into a transfer plasmid for the Bac-Mam system, and prepared recombinant Bac-Mam virus. The obtained virus was amplified in insect Sf9 cells and used to infect mammalian COS-1 cells. Expression of CYP3A4, UGT1A1, and UGT2B7 in COS-1 cell homogenates were confirmed by immunoblotting. Optimum infection conditions including the amount of Bac-Mam virus, culture days before collection, and concentration of sodium butyrate, an enhancer of viral-transduction were determined by monitoring CYP3A4 expression. Expressed CYP3A4 showed appropriate activity without supplying hemin/5-aminolevulinic acid or co-expressing with NADPH-cytochrome P450 reductase. Further, we compared gene transfer efficiency between the Bac-Mam system and an established method using recombinant plasmid and transfection reagent. Our results indicate that the Bac-Mam system can be applied to introduce drug-metabolizing enzyme genes into mammalian cells that are widely used in drug metabolism research. The expressed enzymes are expected to undergo appropriate post-translational modification as they are in mammalian bodies. The Bac-Mam system may thus accelerate pharmacogenetics and pharmacogenomics research.

DAPL1 is a novel regulator of testosterone production in Leydig cells of mouse testis.

Leydig cells in the testes produce testosterone in the presence of gonadotropins. Therefore, male testosterone levels must oscillate within a healthy spectrum, given that elevated testosterone levels augment the risk of cardiovascular disorders. We observed that the expression of death-associated protein-like 1 (DAPL1), which is involved in the early stages of epithelial differentiation and apoptosis, is considerably higher in the testes of sexually mature mice than in other tissues. Accordingly, Dapl1-null mice were constructed to evaluate this variation. Notably, in these mice, the testicular levels of steroidogenic acute regulatory protein (StAR) and serum testosterone levels were significantly elevated on postnatal day 49. The findings were confirmed in vitro using I-10 mouse testis-derived tumor cells. The in vivo and in vitro data revealed the DAPL1-regulated the expression of StAR involving altered transcription of critical proteins in the protein kinase A and CREB/CREM pathways in Leydig cells. The collective findings implicate DAPL1 as an important factor for steroidogenesis regulation, and DAPL1 deregulation may be related to high endogenous levels of testosterone.

LAPTM4α is targeted from the Golgi to late endosomes/lysosomes in a manner dependent on the E3 ubiquitin ligase Nedd4-1 and ESCRT proteins.

Lysosome-associated protein transmembrane 4α (LAPTM4α) is a four transmembrane-spanning protein primarily localized in endosomes and lysosomes and has several putative lysosomal targeting signals at its C-terminal cytoplasmic domain, including tyrosine-based motifs (YxxΦ) and PY motifs (L/PxxY). LAPTM4α has been previously shown to be ubiquitinated by the E3 ubiquitin ligase Nedd4-1 through binding to its PY motifs and sorted to lysosomes, however, the molecular mechanisms underlying the localization of LAPTM4α to endosomes/lysosomes have not yet been fully elucidated. In the present study, we show that LAPTM4α binds Nedd4-1 in a manner dependent on PY motifs, while the PY motifs and Nedd4-1 are not necessarily required for LAPTM4α ubiquitination. The binding of LAPTM4α with Nedd4-1, however, is necessary for an effective sorting of LAPTM4α from the Golgi to late endosomes/lysosomes. An unexpected finding is that LAPTM4α is localized in the lumen, but not in the limiting membrane, of late endosomes, and degraded in lysosomes over time. Interestingly, we further found that siRNA knockdown of endosomal sorting complexes required for transport (ESCRT) components that mediate sorting of ubiquitinated membrane proteins into intralumenal vesicles (ILVs) of endosomes selectively blocks the transport of LAPTM4α to endosomes. Collectively, these results suggest that trafficking of LAPTM4α from the Golgi to endosomes is promoted by the interaction with Nedd4-1, which further requires ESCRT components. Furthermore, our findings highlight a novel function for ESCRT proteins in mediating protein and/or vesicle trafficking from the Golgi to endosomes/lysosomes.

The carboxyl-terminal di-lysine motif is essential for catalytic activity of UDP-glucuronosyltransferase 1A9.

UDP-Glucuronosyltransferase (UGT) is a type I membrane protein localized to the endoplasmic reticulum (ER). UGT has a di-lysine motif (KKXX/KXKXX) in its cytoplasmic domain, which is defined as an ER retention signal. However, our previous study has revealed that UGT2B7, one of the major UGT isoform in human, localizes to the ER in a manner that is independent of this motif. In this study, we focused on another UGT isoform, UGT1A9, and investigated the role of the di-lysine motif in its ER localization, glucuronidation activity, and homo-oligomer formation. Immunofluorescence microscopy indicated that the cytoplasmic domain of UGT1A9 functioned as an ER retention signal in a chimeric protein with CD4, but UGT1A9 itself could localize to the ER in a di-lysine motif-independent manner. In addition, UGT1A9 formed homo-oligomers in the absence of the motif. However, deletion of the di-lysine motif or substitution of lysines in the motif for alanines, severely impaired glucuronidation activity of UGT1A9. This is the first study that re-defines the cytoplasmic di-lysine motif of UGT as an essential peptide for retaining glucuronidation capacity.

HM1.24/BST-2 is constitutively poly-ubiquitinated at the N-terminal amino acid in the cytoplasmic domain.

HM1.24 (also known as BST-2, CD317, and Tetherin) is a type II single-pass transmembrane glycoprotein, which traverses membranes using an N-terminal transmembrane helix and is anchored in membrane lipid rafts via a C-terminal glycosylphosphatidylinositol (GPI). HM1.24 plays a role in diverse cellular functions, including cell signaling, immune modulation, and malignancy. In addition, it also functions as an interferon-induced cellular antiviral restriction factor that inhibits the replication and release of diverse enveloped viruses, and which is counteracted by Vpu, an HIV-1 accessory protein. Vpu induces down-regulation and ubiquitin conjugation to the cytoplasmic domain of HM1.24. However, evidence for ubiquitination site(s) of HM1.24 remains controversial. We demonstrated that HM1.24 is constitutively poly-ubiquitinated at the N-terminal cytoplasmic domain, and that the mutation of all potential ubiquitination sites, including serine, threonine, cysteine, and lysine in the cytoplasmic domain of HM1.24, does not affect the ubiquitination of HM1.24. We further demonstrated that although a GPI anchor is necessary and sufficient for HM1.24 antiviral activities and virion-trapping, the deleted mutant of GPI does not influence the ubiquitination of HM1.24. These results suggest that the lipid raft localization of HM1.24 is not a prerequisite for the ubiquitination. Collectively, our findings demonstrate that the ubiquitination of HM1.24 occurs at the N-terminal amino acid in the cytoplasmic domain and indicate that the constitutive ubiquitination machinery of HM1.24 may differ from the Vpu-induced machinery.

BRCAness as an Important Prognostic Marker in Patients with Triple-Negative Breast Cancer Treated with Neoadjuvant Chemotherapy: A Multicenter Retrospective Study.

Triple-negative breast cancer (TNBC) has several subtypes. The identification of markers associated with recurrence and poor prognosis in patients with TNBC is urgently needed. BRCAness is a set of traits in which BRCA1 dysfunction, arising from gene mutation, methylation, or deletion, results in DNA repair deficiency. In the current study, we evaluated the clinical significance and prognosis of BRCAness in a multicenter retrospective study. Ninety-four patients with TNBC treated with neoadjuvant chemotherapy were enrolled from three university hospitals for this retrospective study. BRCAness was evaluated in 94 core needle biopsy (CNB) specimens prior to neoadjuvant chemotherapy and 49 surgical specimens without pathological complete response (pCR). The samples were assessed using multiplex ligation-dependent probe amplification, and the amplicons were scored. Of the 94 patients, 51 had BRCAness in CNB specimens. There were no significant differences in pCR rates or recurrence between the BRCAness and non-BRCAness groups. Among surgical specimens, the BRCAness group had a significantly shorter recurrence-free survival and overall survival compared with the non-BRCAness group. The BRCAness of surgical specimens was found to be an important marker to predict prognosis in patients with TNBC after neoadjuvant chemotherapy. A clinical trial to assess the clinical impact of carboplatin with BRCAness is planned.

Lysosomal integral membrane protein LGP85 (LIMP-2) is ubiquitinated at the N-terminal cytoplasmic domain.

LGP85/LIMP-2 is a type III transmembrane glycoprotein of lysosomes, which traverses the membrane twice with an N-terminal uncleaved signal sequence and C-terminal hydrophobic domain. In addition to functioning as a receptor for a lysosomal enzyme ß-glucocerebrosidase and for several enteroviruses, LGP85 plays a key role in the biogenesis and maintenance of endosomal/lysosomal compartments (ELCs). Our previous studies have demonstrated that overexpression of rat LGP85 into COS cells results in the enlarged ELCs, from where membrane trafficking is impaired. We show here that rat LGP85 is polyubiquitinated at the N-terminal short cytoplasmic domain that comprises of only three amino acid residues, alanine, arginine, and cysteine. Replacement of either arginine or cysteine with alanine within the N-terminal cytoplasmic domain did not influence the ubiquitination of LGP85, thereby indicating that ubiquitin (Ub) is conjugated to the α-NH2 group of the N-terminal alanine residue. Furthermore, we were able to define a domain necessary for ubiquitination in a region ranging from the amino acids 156 to 255 within the lumenal domain of LGP85. This is the first report showing that the integral lysosomal membrane protein LGP85 is ubiquitinated.

The number of FoxP3-positive tumor-infiltrating lymphocytes in patients with synchronous bilateral breast cancer.

PURPOSE: In breast cancer, FoxP3-positive tumor-infiltrating lymphocytes (FoxP3+ TILs) vary depending on lymph node status, histological grade, and subtype. All these studies have compared the numbers of FoxP3+ TILs among different hosts, but recruitment of FoxP3+ TILs might depend on each individual's immune environment and each tumor's biological characteristics. In the present study, FoxP3+ TIL numbers were investigated in patients with synchronous bilateral breast cancer (SBBC) to determine the factors that affect FoxP3+ TIL recruitment in the same anti-tumor immune environment. METHODS: Patients diagnosed with SBBC who underwent curative surgery at two institutions were enrolled in this study. Patients who underwent primary systemic therapy or who were diagnosed with ductal carcinoma in situ or who had distant metastases at diagnosis were excluded. The average numbers of Foxp3+ TILs were determined from the scores of five high-power microscopic fields (HPF). The associations between Foxp3+ TIL numbers and the clinicopathological features of bilateral breasts in a single individual were examined. RESULTS: Nuclear grade (NG) (p = 0.007) and subtype (p = 0.03), but not size (p = 0.18) and axillary lymph node (p = 0.23) were significantly associated with increase of FoxP3 + TIL numbers by univariate analysis. Further, only NG was a statistically significant clinicopathological factor for change in the number of FoxP3+ TILs by multivariate analysis (p = 0.046) CONCLUSIONS: There was no relationship between FoxP3+ TIL numbers and cancer progression as reflected in tumor size and axillary lymph node in patients with SBBC. Aggressive biological factors, especially high NG, were significantly related to enhanced recruitment of FoxP3+ TILs.

Investigation of the Endoplasmic Reticulum Localization of UDP-Glucuronosyltransferase 2B7 with Systematic Deletion Mutants.

UDP-Glucuronosyltransferase (UGT) plays an important role in the metabolism of endogenous and exogenous compounds. UGT is a type I membrane protein, and has a dilysine motif (KKXX/KXKXX) in its C-terminal cytoplasmic domain. Although a dilysine motif is defined as an endoplasmic reticulum (ER) retrieval signal, it remains a matter of debate whether this motif functions in the ER localization of UGT. To address this issue, we generated systematic deletion mutants of UGT2B7, a major human isoform, and compared their subcellular localizations with that of an ER marker protein calnexin (CNX), using subcellular fractionation and immunofluorescent microscopy. We found that although the dilysine motif functioned as the ER retention signal in a chimera that replaced the cytoplasmic domain of CD4 with that of UGT2B7, UGT2B7 truncated mutants lacking this motif extensively colocalized with CNX, indicating dilysine motif-independent ER retention of UGT2B7. Moreover, deletion of the C-terminal transmembrane and cytoplasmic domains did not affect ER localization of UGT2B7, suggesting that the signal necessary for ER retention of UGT2B7 is present in its luminal domain. Serial deletions of the luminal domain, however, did not affect the ER retention of the mutants. Further, a cytoplasmic and transmembrane domain-deleted mutant of UGT2B7 was localized to the ER without being secreted. These results suggest that UGT2B7 could localize to the ER without any retention signal, and lead to the conclusion that the static localization of UGT results from lack of a signal for export from the ER.

The clinical impact of MRI screening for BRCA mutation carriers: the first report in Japan.

BACKGROUND: There is no consensus on the appropriate surveillance for high-risk women with breast cancer in Japan. We investigated their imaging features and pathological characteristics to build a proper surveillance system for asymptomatic high-risk individuals in the future. METHODS: We retrospectively reviewed 93 female (median age 43 years) BRCA1 and BRCA2 mutation carriers from our institutional clinical database from 2011 to 2017. The study population was composed of 112 breast cancers. Mammography and MRI were reviewed by examiners blinded to patients' clinical history. Final surgical or biopsy histopathology served as the reference standard in all the patients. RESULTS: Fifty-nine breast cancers met selection criteria; of these, 30 were BRCA1-associated tumors, and 29 were BRCA2-associated tumors. Invasive ductal carcinoma was the most prevalent type in both BRCA1 and BRCA2. There were statistically significant differences in phenotype, nuclear grade, and Ki-67 labeling index between BRCA1 and BRCA2 mutation carriers. Additionally, imaging findings on mammography and MRI were statistically different. Tumors in BRCA2 carriers demonstrated mammographic calcifications more frequently, while those in BRCA1 carriers demonstrated a mass or architectural distortion (P < 0.001). Enhancement pattern on MRI also significantly differed between the two subgroups (P = 0.006). The size of MRI-detected lesions was statistically smaller than the size of those detected by other modalities (P = 0.004). CONCLUSIONS: The imaging and histological characteristics of BRCA1/2 mutation carriers were consistent with other countries' studies. MRI-detected lesions were significantly smaller than lesions detected by non-MRI modality. All lesions in BRCA1 mutation carriers could be detected by MRI.

Genetic variations in triple-negative breast cancers undergoing neo-adjuvant chemotherapy.

Aim: Triple negative breast cancer (TNBC) is known as aggressive subtype and have no identified targeted therapies. We examined the relationship of neoadjuvant chemotherapy response to genetic variations of TNBC. Methods: The tumors used in this study were collected from Showa University Hospital, Japan. Thirteen formalin-fixed paraffin-embedded tumors from Japanese TNBC patients who underwent neoadjuvant chemotherapy were used for analysis. Of these, eight surgically resected tumors showed progressive disease and/or recurrence after treatment (PD/REC), and biopsy tissues from five patients showing pathological complete response (pCR) were analyzed. DNA extracted from tissue sample were analyzed. The Miseq system and Trusight Tumor Sequence panel kit were used to sequence 174 amplicons over 82 exons of 26 cancer-related genes to identify genetic mutations. Results: Seven somatic non-synonymous variants were detected in three genes (FOXL2, PIK3CA, and TP53) in all five pCR patients, and six somatic non-synonymous variants in two genes (PTEN and TP53) were detected in six of eight PD/REC patients. Eight of 13 TNBC tumors were found to have TP53 pathogenic variants, in both pCR and PD/REC cases. Conclusion: Although TP53 variation was detected in both pCR and PD/REC cases, each location and type of the variant were different. We could not identify genetic mutations associated with chemotherapy response and recurrence.

Raman spectroscopy for the diagnosis of unlabeled and unstained histopathological tissue specimens.

AIM: To investigate the possibility of diagnosing gastric cancer from an unstained pathological tissue using Raman spectroscopy, and to compare the findings to those obtained with conventional histopathology. METHODS: We produced two consecutive tissue specimens from areas with and without cancer lesions in the surgically resected stomach of a patient with gastric cancer. One of the two tissue specimens was stained with hematoxylin and eosin and used as a reference for laser irradiation positioning by the spectroscopic method. The other specimen was left unstained and used for Raman spectroscopy analysis. RESULTS: A significant Raman scattering spectrum could be obtained at all measurement points. Raman scattering spectrum intensities of 725 cm-1 and 782 cm-1, are associated with the nucleotides adenine and cytosine, respectively. The Raman scattering spectrum intensity ratios of 782 cm-1/620 cm-1, 782 cm-1/756 cm-1, 782 cm-1/1250 cm-1, and 782 ​​cm-1/1263 cm-1 in the gastric adenocarcinoma tissue were significantly higher than those in the normal stomach tissue. CONCLUSION: The results of this preliminary experiment suggest the feasibility of our spectroscopic method as a diagnostic tool for gastric cancer using unstained pathological specimens.

The association between p16 and Ki-67 immunohistostaining and the progression of cervical intraepithelial neoplasia grade 2.

OBJECTIVE: To compare the frequency of p16- and Ki-67-positive cells on immunohistostaining among women with progression, persistence, or regression of cervical intraepithelial neoplasia grade 2 (CIN2). METHODS: A retrospective study was conducted of women with CIN2 diagnosed by histology who were treated at a university hospital in Japan during 2004-2011. The immunostaining patterns for p16 and Ki-67 were analyzed and compared between patients with disease progression, persistence, or regression. Kaplan-Meier analysis was used to evaluate the progression rates stratified by immunostaining, and multivariate analysis of risk factors for progression was performed using the Cox proportional hazards model. RESULTS: The analysis included 59 women with progression, 35 women with persistence, and 28 women with regression. Deep p16 expression (staining in more than half of the cervical intraepithelial compartment) and positive Ki-67 staining in more than 50% of cells were significantly more common among women with progression than among those with regression. The risk factors for progression of CIN2 were deep p16 expression (P<0.001) and a Ki-67 ratio of more than 50% (P<0.001). CONCLUSION: Among women with CIN2, positive immunohistostaining for p16 and Ki-67 was strongly associated with disease progression.

Cytology of low-grade cribriform cystadenocarcinoma in salivary glands: Cytological and immunohistochemical distinctions from other salivary gland neoplasms.

Low-grade cribriform cystadenocarcinoma of the parotid gland is rare malignancy that is classified as a variant of cystadenocarcinoma. In routine cytologic slides from fine-needle aspiration of a parotid gland, we found several pseudopapillary clusters comprising mucus-producing cells. They included a few tumor cells having three-dimensional nuclear atypia and slight hyperchromatism, although most of the tumor cells showed bland nuclei. Our initial cytological diagnosis was: "Indeterminate. Uncertain whether cystadenocarcinoma or cystadenoma." The subsequent histological diagnosis was low-grade cribriform cystadenocarcinoma. Immunohistochemical staining showed diffuse and strong reactivity for S-100; tumor nests that were rimmed by p63(+) cells, which suggests intraductal proliferation. Here, we report cytomorphological findings of this case, and discuss cytological and immunohistochemical distinctions between low-grade cribriform cystadenocarcinoma and other salivary gland tumors, including a review of the literature.

Intracellular Dynamics and Fate of a Humanized Anti-Interleukin-6 Receptor Monoclonal Antibody, Tocilizumab.

Tocilizumab (TCZ), a humanized anti-interleukin-6 (IL-6) receptor (IL-6R) monoclonal antibody, abrogates signal transducer protein gp130-mediated IL-6 signaling by competitively inhibiting the binding of IL-6 to the receptor, and shows clinical efficacy in autoimmune and inflammatory diseases. Despite accumulating evidence for therapeutic efficacy, the behavior and fate of TCZ at the cellular level remain largely unknown. To address this, we evaluated the endocytosis and intracellular trafficking of IL-6R in HeLa cells. The results of our study provide evidence that IL-6R is constitutively internalized from the cell surface by ligand or TCZ binding and the expression of gp130 in an independent manner and is targeted via endosomes without being significantly directed to the recycling pathway to, and degraded in, lysosomes. Furthermore, the cytoplasmic tail of IL-6R is required for constitutive endocytosis of the receptor, which is mediated by the clathrin and AP-2 complex. We further demonstrate that FcRn, whose function is to regulate the serum persistence of IgG, is confined primarily to early/recycling endosomes and rapidly transits between these compartments and late endosomes/lysosomes without being degraded. Importantly, the expression of FcRn induces the segregation of TCZ from IL-6R, resulting in extensive colocalization of TCZ and FcRn in IL-6R-depleted endosomal compartments. Collectively, our results suggest that FcRn can accelerate the retrieval of the internalized TCZ, not only from endosomes but also from lysosomes. Our findings provide new insight into the mechanism by which the antibody internalized into cells is rescued from lysosomal degradation and into how its serum levels are maintained.

Mitophagy is primarily due to alternative autophagy and requires the MAPK1 and MAPK14 signaling pathways.

In cultured cells, not many mitochondria are degraded by mitophagy induced by physiological cellular stress. We observed mitophagy in HeLa cells using a method that relies on the pH-sensitive fluorescent protein Keima. With this approach, we found that mitophagy was barely induced by carbonyl cyanide m-chlorophenyl hydrazone treatment, which is widely used as an inducer of PARK2/Parkin-related mitophagy, whereas a small but modest amount of mitochondria were degraded by mitophagy under conditions of starvation or hypoxia. Mitophagy induced by starvation or hypoxia was marginally suppressed by knockdown of ATG7 and ATG12, or MAP1LC3B, which are essential for conventional macroautophagy. In addition, mitophagy was efficiently induced in Atg5 knockout mouse embryonic fibroblasts. However, knockdown of RAB9A and RAB9B, which are essential for alternative autophagy, but not conventional macroautophagy, severely suppressed mitophagy. Finally, we found that the MAPKs MAPK1/ERK2 and MAPK14/p38 were required for mitophagy. Based on these findings, we conclude that mitophagy in mammalian cells predominantly occurs through an alternative autophagy pathway, requiring the MAPK1 and MAPK14 signaling pathways.

BRCAness predicts resistance to taxane-containing regimens in triple negative breast cancer during neoadjuvant chemotherapy.

BACKGROUND: To provide optimal treatment of heterogeneous triple negative breast cancer (TNBC), we need biomarkers that can predict the chemotherapy response. PATIENTS AND METHODS: We retrospectively investigated BRCAness in 73 patients with breast cancer who had been treated with taxane- and/or anthracycline-based neoadjuvant chemotherapy (NAC). Using multiplex, ligation-dependent probe amplification on formalin-fixed core needle biopsy (CNB) specimens before NAC and surgical specimens after NAC. BRCAness status was assessed with the assessor unaware of the clinical information. RESULTS: We obtained 45 CNB and 60 surgical specimens from the 73 patients. Of the 45 CNB specimens, 17 had BRCAness (38.6% of all subtypes). Of the 23 TNBC CNB specimens, 14 had BRCAness (61% of TNBC cases). The clinical response rates were significantly lower for BRCAness than for non-BRCAness tumors, both for all tumors (58.8% vs. 89.3%, P = .03) and for TNBC (50% vs. 100%, P = .02). All tumors that progressed with taxane therapy had BRCAness. Of the patients with TNBC, those with non-BRCAness cancer had pathologic complete responses significantly more often than did those with BRCAness tumors (77.8% vs. 14.3%, P = .007). After NAC, the clinical response rates were significant lower for BRCAness than for non-BRCAness tumors in all subtypes (P = .002) and in TNBC cases (P = .008). After a median follow-up of 26.4 months, 6 patients-all with BRCAness-had developed recurrence. Patients with BRCAness had shorter progression-free survival than did those with non- BRCAness (P = .049). CONCLUSION: Identifying BRCAness can help predict the response to taxane, and changing regimens for BRCAness TNBC might improve patient survival. A larger prospective study is needed to further clarify this issue.