Proton Pump Inhibitors and Cyclin-Dependent Kinase 4/6 Inhibitors in Patients With Breast Cancer.

BACKGROUND: Proton pump inhibitors (PPIs) reduce the bioavailability of several anticancer drugs. The impact of PPIs co-administered with cyclin-dependent kinase 4 and 6 inhibitors is controversial. We aimed to clarify whether the concomitant use of PPIs impacts palbociclib and abemaciclib effectiveness in breast cancer treatment. PATIENTS AND METHODS: This multicenter, retrospective, observational study, conducted across 4 medical institutions in Japan, consecutively included patients with endocrine-resistant metastatic breast cancer, receiving palbociclib or abemaciclib between December 2017 and August 2022. Propensity score-matched analyses were performed. Treatment efficacy and safety with and without PPIs were compared. Progression-free survival and overall survival were estimated using the Kaplan-Meier method and compared using a log-rank test. A Cox proportional hazards model was used to estimate the hazard ratio. RESULTS: The study included 240 patients. After 1:1 matching, 112 patients were treated with and without PPIs. The median progression-free survival period was 1.2 years in the PPI group and 1.3 years in the non-PPI group (hazard ratio, 1.19; 95% CI, 0.70-2.02). The median overall survival period was 3.6 years in the PPI group, whereas it was not reached in the non-PPI group (hazard ratio, 1.23; 95% CI, 0.61-2.47). Consistent results were obtained for subgroups receiving palbociclib (n = 177) and abemaciclib (n = 63) without propensity score matching. Adverse event incidence and severity were similar in both groups. CONCLUSION: The effectiveness of cyclin-dependent kinase 4/6 inhibitors is unlikely to be affected by concomitant PPI use. Future prospective pharmacokinetic studies are warranted.

Application of an artificial intelligence-based system in the diagnosis of breast ultrasound images obtained using a smartphone.

BACKGROUND: Breast ultrasound (US) is useful for dense breasts, and the introduction of artificial intelligence (AI)-assisted diagnoses of breast US images should be considered. However, the implementation of AI-based technologies in clinical practice is problematic because of the costs of introducing such approaches to hospital information systems (HISs) and the security risk of connecting HIS to the Internet to access AI services. To solve these problems, we developed a system that applies AI to the analysis of breast US images captured using a smartphone. METHODS: Training data were prepared using 115 images of benign lesions and 201 images of malignant lesions acquired at the Division of Breast Surgery, Gifu University Hospital. YOLOv3 (object detection models) was used to detect lesions on US images. A graphical user interface (GUI) was developed to predict an AI server. A smartphone application was also developed for capturing US images displayed on the HIS monitor with its camera and displaying the prediction results received from the AI server. The sensitivity and specificity of the prediction performed on the AI server and via the smartphone were calculated using 60 images spared from the training. RESULTS: The established AI showed 100% sensitivity and 75% specificity for malignant lesions and took 0.2 s per prediction with the AI sever. Prediction using a smartphone required 2 s per prediction and showed 100% sensitivity and 97.5% specificity for malignant lesions. CONCLUSIONS: Good-quality predictions were obtained using the AI server. Moreover, the quality of the prediction via the smartphone was slightly better than that on the AI server, which can be safely and inexpensively introduced into HISs.

Oncofertility-related psycho-educational therapy for young adult patients with breast cancer and their partners: Randomized controlled trial.

BACKGROUND: A couples' psycho-educational program called Oncofertility! Psycho-Education and Couple Enrichment (O!PEACE) therapy was created and its effect when provided before cancer treatment was examined. METHODS: This multicenter randomized controlled trial with nonmasking, parallel two-group comparison enrolled women aged 20 to 39 years with early-stage breast cancer and their partners. They were randomly assigned to receive O!PEACE (37 couples) or usual care (37 couples). Primary end points were cancer-related posttraumatic stress symptoms, symptoms of depression, and anxiety. Secondary end points were stress-coping strategies, resilience, and marital relationship. RESULTS: Women receiving psycho-educational therapy had significantly reduced Impact of Event Scale-revised version for Japanese scores (p = .011, ηp 2  =  = .089). For patients with Impact of Event Scale-revised version for Japanese scores at baseline ≥18.27, O!PEACE therapy improved these scores when compared with usual care (U = 172.80, p = .027, r = 0.258). A >5-point reduction was present in 59.3% and 30% of women in the O!PEACE therapy and usual-care groups, respectively. For partners, O!PEACE therapy significantly improved stress-coping strategies (95% CI, -0.60 to -0.05; p = .018, ηp 2  =  = .074) and escape-avoidance marital communication (95% CI, -0.33 to -0.08; p = .001, ηp 2  = .136). O!PEACE therapy significantly improved the partners' support (95% CI, 0.10-0.50; p = .001, ηp 2  = .127), the rate of receiving fertility preservation consultations, and knowledge levels. CONCLUSIONS: O!PEACE therapy before cancer treatment can improve posttraumatic stress symptoms, stress-coping behavior, and marital relationships. Larger sample sizes and longer term follow-up are required. PLAIN LANGUAGE SUMMARY: A psycho-educational program, the Oncofertility! Psycho-Education and Couple Enrichment (O!PEACE) therapy program was developed and evaluated for women diagnosed with breast cancer and their partners. A multicenter randomized controlled trial showed that the O!PEACE psycho-educational therapy, with only two precancer treatment sessions, can reduce cancer-related posttraumatic stress symptoms and improve oncofertility knowledge and marital relationships in young adult patients with breast cancer. The therapy could also improve stress-coping strategies in marital communications with their partners. Couples may use O!PEACE psycho-educational therapy to consider fertility preservation and improve their psychosocial aspects.

Gastric carcinosarcoma with FGFR2 amplification under long-term control with pazopanib: a case report and literature review.

BACKGROUND: Gastric carcinosarcoma is most frequently diagnosed at an advanced stage when the tumor is generally large with invasion into other organs, lymph node metastasis, and distant metastasis. Standard chemotherapy has not been established, and surgery is the only curative treatment. Here, we present a case of postoperative recurrence of gastric carcinosarcoma under long-term tumor control with pazopanib. CASE PRESENTATION: A 77-year-old man was referred to our hospital because of nausea and vomiting. Computed tomography and upper gastrointestinal endoscopy revealed a type 1 tumor arising from the gastric antrum and extending into the duodenal bulb. He underwent distal gastrectomy (D2) with Roux-en-Y reconstruction. Histopathologically, the tumor had mixed adenocarcinoma and sarcoma components. According to the tumor-node-metastasis classification, the diagnosis was primary gastric carcinosarcoma pT1bN1M0 stage IB. Liver metastasis was detected 2 months after surgery; multiple lung metastases were detected 17 month after surgery. A genomic profiling test was performed using liver specimens as the patient became refractory to chemotherapy commonly used for gastric cancer, and the test revealed FGFR2 amplification along with TP53 R209*, AKT3 N127D, NOTCH1 A2036T, and POLD1 M161I. The patient was treated with pazopanib (800 mg/daily), and the tumor growth was controlled for 11 months. CONCLUSIONS: We report a case of postoperative recurrence of gastric carcinosarcoma under long-term tumor control with pazopanib. This case suggested that pazopanib may be effective in treating gastric carcinosarcoma.

Current status of AYA-generation breast cancer: trends worldwide and in Japan.

Breast cancer (BC) is the most common cancer worldwide among women. In Japan, the incidence of BC gradually increased. The recent number of adolescent and young adult (AYA)-BC patients is approximately 4,000-5,000 every year, accounting for 5% of all BC cases. BC in young people has been attracting attention since Japan's third basic plan to promote cancer control programs incorporated cancer control measures for pediatric (age ≤ 14 years) and adolescent and young adult (AYA)-generation (age 15-39 years) cancers in 2018. Attention is needed to detect AYA-BC because of the presence of dense breasts. AYA-BC patients are clinically characterized by larger tumor size, more lymph node metastases, advanced stages, and a higher rate of aggressive phenotypes, such as triple-negative or HER2-positive subtypes, and are strongly associated with family history and genetic germline alterations, including hereditary breast and ovarian cancers. Given that AYA-BC patients show a poorer prognosis than older BC patients, they often require intensive therapies, including surgery, radiation, chemotherapy, and endocrine therapy. We must solve many survivorship-associated problems in AYA-BC patients, including fertility preservation, comorbidity after treatment, and long-term follow-up. Under these circumstances, national and local governments and various academic societies have started addressing these problems by formulating laws and guidelines, establishing medical systems, and offering financial support to conquer cancer and maintain a better quality of life. This review summarizes the current trends of AYA-BC worldwide and in Japan. Further Japan-specific data on AYA-BC are required to clarify its characteristics and improve prognosis and survivorship.

p53/Mieap-regulated mitochondrial quality control plays an important role as a tumor suppressor in gastric and esophageal cancers.

Mitochondria-eating protein (Mieap) plays a critical role in mitochondrial quality control (MQC) and functions as a p53-inducible tumor suppressor. This study aimed to examine its role in gastric cancer (GC) and esophageal cancer (EC). GC cells were infected with Mieap-overexpressing adenovirus (Ad-Mieap) and subjected to fluorescence-activated cell sorting (FACS), western blotting, and caspase assays. Thereafter, we evaluated the potential disruption of the p53/Mieap-regulated MQC pathway in vivo. Methylation-specific PCR (MSP) for Mieap, NIX, and BNIP3 promoters was performed and p53 mutations were detected using cryopreserved surgical specimens. Exogenous Mieap in GC cells induced the formation of vacuole-like structures (called MIVs, Mieap-induced vacuoles) and caspase-dependent cell death, with the activation of both caspase-3 and caspase-9. Of the 47 GC patients, promoter methylation in Mieap, BNIP3, and NIX was identified in two (4.3%), 29 (61.7%), and zero (0%) specimens, respectively. In total, 33 GC patients (70.2%) inactivated this MQC pathway. Amazingly, BNIP3 promoter in the normal epithelium was highly methylated in 18 of the 47 GC patients (38.3%). In EC patients, this MQC pathway was also inactivated in ten of 12 patients (83.3%). These results indicate that p53/Mieap-regulated MQC plays an important role in upper gastrointestinal (GI) tumor suppression, possibly, in part, through the mitochondrial apoptotic pathway.

Intratumoral Adipocyte-High Breast Cancer Enrich for Metastatic and Inflammation-Related Pathways but Associated with Less Cancer Cell Proliferation.

Cancer-associated adipocytes are known to cause inflammation, leading to cancer progression and metastasis. The clinicopathological and transcriptomic data from 2256 patients with breast cancer were obtained based on three cohorts: The Cancer Genome Atlas (TCGA), GSE25066, and a study by Yau et al. For the current study, we defined the adipocyte, which is calculated by utilizing a computational algorithm, xCell, as "intratumoral adipocyte". These intratumoral adipocytes appropriately reflected mature adipocytes in a bulk tumor. The amount of intratumoral adipocytes demonstrated no relationship with survival. Intratumoral adipocyte-high tumors significantly enriched for metastasis and inflammation-related gene sets and are associated with a favorable tumor immune microenvironment, especially in the ER+/HER2- subtype. On the other hand, intratumoral adipocyte-low tumors significantly enriched for cell cycle and cell proliferation-related gene sets. Correspondingly, intratumoral adipocyte-low tumors are associated with advanced pathological grades and inversely correlated with MKI67 expression. In conclusion, a high amount of intratumoral adipocytes in breast cancer was associated with inflammation, metastatic pathways, cancer stemness, and favorable tumor immune microenvironment. However, a low amount of adipocytes was associated with a highly proliferative tumor in ER-positive breast cancer. This cancer biology may explain the reason why patient survival did not differ by the amount of adipocytes.

High Expression of miR-34a Associated with Less Aggressive Cancer Biology but Not with Survival in Breast Cancer.

Most breast cancer (BC) patients succumb to metastatic disease. MiR-34a is a well-known tumor suppressive microRNA which exerts its anti-cancer functions by playing a role in p53, apoptosis induction, and epithelial-mesenchymal transition (EMT) suppression. Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) cohorts were used to test our hypothesis that miR-34a high BCs translate to less aggressive cancer biology and better survival in large cohorts. There was no association between miR-34a expression levels and clinicopathological features of BC patients except for HER2 positivity. MiR-34a high expressing tumors were associated with lower Nottingham pathological grades and lower MKI67 expression. In agreement, high miR-34a tumors demonstrated lower GSVA scores of cell cycle and cell proliferation-related gene sets. High miR-34a tumors enriched the p53 pathway and apoptosis gene sets. Unexpectedly, high miR-34a tumors also associated with elevated EMT pathway score and ZEB1 and two expressions. MiR-34a expression did not associate with any distant metastasis. Further, high miR-34a tumors did not associate with better survival compared with miR-34a low tumors. In conclusion, the clinical relevance of miR-34a high expressing tumors was associated with suppressed cell proliferation, enhanced p53 pathway and apoptosis, but enhanced EMT and these findings did not reflect better survival outcomes in large BC patient cohorts.

High Expression of microRNA-143 is Associated with Favorable Tumor Immune Microenvironment and Better Survival in Estrogen Receptor Positive Breast Cancer.

microRNA-143(miR-143) is a well-known tumor suppressive microRNA that exhibits anti-tumoral function by targeting KRAS signaling pathways in various malignancies. We hypothesized that miR-143 suppresses breast cancer progression by targeting KRAS and its effector molecules. We further hypothesized that high expression of miR-143 is associated with a favorable tumor immune microenvironment of estrogen receptor (ER)-positive breast cancer patients which result in improved survival. Two major publicly available breast cancer cohorts; The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) were used. The miR-143 high expression group was associated with increased infiltration of anti-cancer immune cells and decreased pro-cancer immune cells, as well as enrichment of the genes relating to T helper (Th1) cells resulting in improved overall survival (OS) in ER-positive breast cancer patients. To the best of our knowledge, this is the first study to demonstrate that high expression of miR-143 in cancer cells associates with a favorable tumor immune microenvironment, upregulation of anti-cancer immune cells, and suppression of the pro-cancer immune cells, associating with better survival of the breast cancer patients.

The inhibition of thymidine phosphorylase can reverse acquired 5FU-resistance in gastric cancer cells.

BACKGROUND: 5FU can be converted to its active metabolite fluoro-deoxyuridine monophosphate (FdUMP) through two pathways: the orotate phosphoribosyl transferase-ribonucleotide reductase (OPRT-RR) pathway and the thymidine phosphorylase-thymidine kinase (TP-TK) pathway. We investigated the mechanism underlying 5FU-resistance, focusing on the changes in the 5FU metabolisms. METHODS: MKN45 and 5FU-resistant MKN45/F2R cells were treated with 5FU or fluoro-deoxyuridine (FdU) in combination with hydroxyurea (HU) or tipiracil (TPI). The amount of FdUMP was determined by the density of the upper band of thymidylate synthase on Western blotting. RESULTS: The MKN45/F2R cells exhibited 5FU resistance (37.1-fold) and showed decreased OPRT and increased TP levels. In both cells, the FdUMP after treatment with 5FU was decreased when RR was inhibited by HU but not when TP was inhibited by TPI. A metabolome analysis revealed the loss of intracellular deoxyribose 1-phosphate (dR1P) in both cells, indicating that FdUMP was synthesized from 5FU only through the OPRT-RR pathway because of the loss of dR1P. After the knockdown of TK, the FdUMP after treatment with FdU was decreased in MKN45 cells. However, it was not changed in MKN45/F2R cells. Furthermore, TP inhibition caused an increase in FdUMP after treatment with 5FU or FdU and reversed the 5FU resistance in MKN45/F2R cells, indicating that FdUMP was reduced through the TP-TK pathway in MKN45/F2R cells. CONCLUSIONS: In MKN45/F2R cells, the reduction of FdUMP through the TP-TK pathway caused 5FU resistance, and the inhibition of TP reversed the resistance to 5FU, suggesting that the combination of 5FU and TPI is a promising cancer therapy.

Synthetic miR-143 Inhibits Growth of HER2-Positive Gastric Cancer Cells by Suppressing KRAS Networks Including DDX6 RNA Helicase.

Gastric cancer (GC) is one of the most common cancers worldwide. In the clinical setting, the identification of HER2 overexpression in GC was a significant finding, as trastuzumab, an anti-HER2 drug, provides a survival advantage to HER2-positive GC patients. In HER2-postive GC, the dysregulation of PI3K/AKT and MAPK/ERK signaling pathways has been reported, and inhibition of these pathways is an important therapeutic strategy. MiR-143 is known to act as a tumor suppressor in several cancers, such as bladder cancer, breast cancer, colorectal cancer, and gastric cancer. In the current study, we developed a novel chemically-modified miR-143 and explored the functions of this synthetic miR-143 (syn-miR-143) in HER2-positive gastric cancer. The expression level of miR-143 was down-regulated in GC cell lines, including HER2-positive GC cell lines, MKN7, and KATO-III. The ectopic expression of miR-143 in those cell lines suppressed cell growth through systemic silencing of KRAS and its effector signaling molecules, AKT and ERK. Furthermore, syn-miR-143 indirectly down-regulated the expression of HER2, an upstream molecule of KRAS, through silencing DEAD/H-box RNA helicase 6 (DDX6), RNA helicase, which enhanced HER2 protein expression at the translational step in HER2-positive GC cells. These findings suggested that syn-miR-143 acted as a tumor suppressor through the impairment of KRAS networks including the DDX6.

Possible role of p53/Mieap-regulated mitochondrial quality control as a tumor suppressor in human breast cancer.

Mitochondria-eating protein (Mieap), encoded by a p53-target gene, plays an important role in mitochondrial quality control (MQC). Mieap has been reported to have a critical role in tumor suppression in colorectal cancer. Here, we investigated its role as a tumor suppressor in breast cancer. The enforced expression of exogenous Mieap in breast cancer cells induced caspase-dependent apoptosis, with activation of both caspase-3/7 and caspase-9. Immunohistochemistry revealed endogenous Mieap in the cytoplasm in 24/75 (32%) invasive ductal carcinomas (IDC), 15/27 (55.6%) cases of ductal carcinoma in situ (DCIS) and 16/18 (88.9%) fibroadenomas (FA) (IDC vs DCIS; P = 0.0389, DCIS vs FA; P = 0.0234, IDC vs FA; P < 0.0001). In IDC, the Mieap promoter was methylated in 6/46 (13%) cases, whereas p53 was mutated in 6/46 (13%) cases. Therefore, the p53/Mieap-regulated MQC pathway was inactivated in 12/46 IDC (26.1%). Interestingly, all tumors derived from the 12 patients with Mieap promoter methylation or p53 mutations pathologically exhibited more aggressive and malignant breast cancer phenotypes. Impairment of p53/Mieap-regulated MQC pathway resulted in significantly shorter disease-free survival (DFS) (P = 0.021), although p53 status is more prognostic in DFS than Mieap promoter methylation. These results indicate that p53/Mieap-regulated MQC has a critical role in tumor suppression in breast cancer, possibly in part through mitochondrial apoptotic pathway.

Oncogene RNA helicase DDX6 promotes the process of c-Myc expression in gastric cancer cells.

Human DEAD-box RNA helicase gene DDX6 was cloned from B-cell lymphoma cell line RC-K8. Previously, we reported that DDX6 acts as oncogene in several cancers such as colorectal cancer and hepatocellular carcinoma. However, the detailed mechanism of DDX6 action in carcinogenesis is largely unknown. In this study, we examined the functions of DDX6 in clinical gastric cancer (GC) samples and GC cells. DDX6 protein expression levels of cancer samples were higher than those of the adjacent normal tissues in 25 clinical GC samples (median value: 1.4 times higher). Also, the results of an RNA immunoprecipitation-assay (RIP-assay) showed that DDX6 associated with c-Myc mRNA. Moreover, enforced overexpression of DDX6 promoted both mRNA and protein expression of c-Myc in GC cells. On the other hand, the gene silencing of DDX6 induced growth suppression through down-regulation of c-Myc in GC cells grown in either two or three dimensions. Furthermore, c-Myc mRNA expression levels of cancer samples were higher than those of the adjacent normal tissues in DDX6 up-regulated-GC clinical samples. Our findings in this study suggested that DDX6 acted as oncogene in GC cells through promotion of c-Myc expression by association with the mRNA of c-Myc.

The mode of progressive disease affects the prognosis of patients with metastatic breast cancer.

BACKGROUND: According to the Response Evaluation Criteria in Solid Tumors (RECIST), progressive disease (PD) is diagnosed under two conditions: an increase in size of pre-existing lesions (IS) and the appearance of new lesions (NL). We retrospectively investigated the difference in the prognosis between IS and NL. METHODS: Patients receiving drug therapies for metastatic breast cancer between 2004 and 2015 at our institution were reviewed. The survival time after NL and IS was compared and the frequency of NL with each drug calculated. RESULTS: For the 107 eligible patients, the survival time after NL at second-line chemotherapy was significantly worse than after IS (median survival time 4.3 months vs. 20.3 months, p = 0.0048). Maintenance therapy with bevacizumab or trastuzumab had a high frequency of NL (88.9%), and third-line eribulin had a low frequency of NL (16.7%). A multivariate analysis showed that NL at second-line chemotherapy was not an independent risk factor (hazard ratio 1.02, 95%; confidence interval 0.54-1.93, p = 0.95) for the total survival time. CONCLUSIONS: Patients with IS had a better survival after PD than those with NL. We may be able to avoid changing drug therapy for patients without NL and allow them to continue drug therapy for longer.

DEAD-Box Protein RNA-Helicase DDX6 Regulates the Expression of HER2 and FGFR2 at the Post-Transcriptional Step in Gastric Cancer Cells.

The human DEAD/H-box RNA helicase DDX6 (RCK/p54) is a protein encoded by the fusion gene from the t(11;14)(q23;q32) chromosomal translocation observed in human B-cell lymphoma cell line RC-K8. DDX6 has a variety of functions such as translation initiation, pre-mRNA splicing, and ribosome assembly. However, details of the regulatory mechanism governing DDX6 and the functions of DDX6 are largely unknown. Previously, we reported that DDX6 is overexpressed in most malignant cell lines and clinical colorectal tumor samples and that DDX6 positively contributes to the pathogenesis of various cancers. In the current study, we aimed at revealing the function of DDX6 in HER2 and FGFR2 related human gastric cancer (GC) by using clinical samples and GC cell lines. DDX6 protein was overexpressed in about 60% of the clinical samples; HER2, in 35%; and FGFR2, in 30%, (n = 20). Interestingly, the DDX6 protein was overexpressed in all HER2-positive samples (n = 7), and in 83% (5 of 6) of the FGFR2-positive samples, which could reflect the contribution of DDX6 to the expression of HER2 and FGFR2. In the GC cell line MKN7, which has HER2 amplification, the knockdown of DDX6 by siR-DDX6 led to the decreased expression of the HER2 protein. On the other hand, the knockdown of HER2 did not influence the DDX6 expression. Similar results were also obtained for the KATO-III and HSC39 cell lines having amplified FGFR2 expression. The increased expression of DDX6 induced a significantly increased expression of the HER2 protein without increasing the mRNA expression. The results of an RNP Immunoprecipitation (RIP)-assay using GC cells indicated that the DDX6 protein acted as an RNA-binding protein for HER2 and FGFR2 mRNAs and positively regulated their post-transcriptional processes. These findings demonstrated that DDX6 was an upstream molecule that positively regulated the expression of HER2 and FGFR2 at the post-transcriptional step in GC cells.

The diagnosis of a metastatic breast tumor from ovarian cancer by the succession of a p53 mutation: a case report.

BACKGROUND: Metastatic breast tumors from other organs are very rare. We herein describe the case of a patient with a metastatic breast tumor due to ovarian cancer who was diagnosed by the succession of a p53 mutation. CASE PRESENTATION: The patient was a 59-year-old woman with sigmoid colon stenosis. Diagnostic imaging revealed a pelvic mass, multiple liver tumors, ascites, and multiple swollen para-aortic lymph nodes, suggesting an advanced ovarian tumor. Transverse loop colostomy and partial resection of the greater omentum was performed followed by six cycles of paclitaxel with carboplatin chemotherapy (TC therapy). Her cancer almost disappeared, with the exception of a small tumor in her pelvis. Simple hysterectomy with bilateral salpingo-oophorectomy was performed. Two years and 5 months after the second surgery, a mass was detected in her right breast and simple mastectomy was performed. A histological examination of the tumors from the first surgery revealed infiltrating papillary adenocarcinoma and the solid nest proliferation of atypical cells with comedo necrosis and psammoma bodies. The findings of an immunohistochemical analysis were as follows: cancer antigen 125 (CA125 (+)), cytokeratin 7 (CK7 (+)), cytokeratin 20 (CK20 (-)), p53 (+) and CDX2 (-), estrogen receptor (ER (slightly +)), progesterone receptor (PR (slightly +)), and human epidermal growth factor receptor 2 (HER2 (1+)). The breast tumors presented similar morphological features (ER (-), PR (-), HER2 (-), CA125 (+), CK7 (+), CK20 (-), p53 (+), mammaglobin (-), and GCDFP15 (-)), which were not characteristic of breast cancer. A direct sequencing analysis of p53 revealed a p.V173M mutation in exon 5 in both the breast tumor and the ovarian cancer. It was not detected in normal tissue, suggesting that the breast tumors were metastatic serous adenocarcinomas from ovarian cancer. CONCLUSIONS: A direct sequencing mutation analysis of p53 was useful for distinguishing the primary tumor from the metastatic tumor. We should resect metastatic breast tumors to the extent that is possible because the prognosis of such patients is relatively good.

Positive feedback of DDX6/c-Myc/PTB1 regulated by miR-124 contributes to maintenance of the Warburg effect in colon cancer cells.

The human DEAD/H-box RNA helicase gene DDX6 is a target of the t(11;14)(q23;q32) chromosomal translocation observed in human B-cell lymphoma, and the overexpression of its protein has been shown to cause malignant transformation. DDX6 has a variety of functions such as translation initiation, pre-mRNA splicing, ribosome assembly, and more. However, details of the regulatory mechanism of DDX6 and functions of DDX6 in cancer cells are largely unknown. On the other hand, the Warburg effect is a well-known feature of cancer cells. Pyruvate kinase in muscle (PKM), which is a rate-limiting glycolytic enzyme, has 2 isoforms, PKM1 and PKM2. It has been frequently reported that PKM2 is a tumor-specific isoform and promotes the Warburg effect. However, the functions of the PKM1 gene have been hardly mentioned. Here, we showed that DDX6 was overexpressed in colorectal cancer specimens and regulated by microRNA (miR)-124 in colon cancer cells. Also, a DDX6/c-Myc/PTB1 positive feedback circuit regulated by miR-124 was shown to be established and to contribute to maintenance of the Warburg effect. Moreover, we showed that knockdown of DDX6 induced mainly apoptosis through an imbalance of PKM gene expression, especially causing down-regulation of PKM1 in colon cancer cells. These results suggest that miR-124 is a fine tuner of the Warburg effect and that DDX6 is one of the key molecules in Warburg effect-related miR-124 targeting various genes.

Combination therapy with zoledronic acid and cetuximab effectively suppresses growth of colorectal cancer cells regardless of KRAS status.

Targeted molecular therapy is an effective anticancer strategy. Anti-EGFR monoclonal antibodies such as cetuximab (CTX) have been approved for the treatment of various malignancies, including colorectal cancer (CRC) with wild-type KRAS. However, their efficacy in patients with KRAS mutations has not been established. Therefore, we investigated whether CTX treatment was effective as a single agent or in combination with zoledronic acid (ZOL) in human CRC cell lines with different KRAS status. CRC cell lines SW48 (wild-type KRAS) and LS174T (mutant KRAS) were treated with ZOL, CTX and a combination of both drugs. Cytotoxicity was measured using the MTT assay. Changes in the levels of intracellular signaling proteins were evaluated using western blot analysis. Finally, we evaluated the efficacy of the combination treatment in an in vivo xenograft model. We observed that ZOL apparently inhibited growth in both cell lines, whereas CTX showed little effect. ZOL also increased the levels of unprenylated RAS. Combined ZOL and CTX treatment was synergistic in both cell lines and was associated with inhibition of the RAS-MAPK and AKT-mTOR signaling pathways. Furthermore, the combination treatment was more effective in suppressing the growth of xenografts derived from both SW48 and LS174T cells; this effect was associated with increased apoptosis. These results demonstrate that ZOL inhibits the growth of colon cancer cells regardless of KRAS status, and combination therapy using ZOL and CTX enhances this growth suppression. These findings suggest a novel strategy for the treatment of CRC independent of KRAS mutational status.

MiR-133b inhibits growth of human gastric cancer cells by silencing pyruvate kinase muscle-splicer polypyrimidine tract-binding protein 1.

The metabolism in tumor cells shifts from oxidative phosphorylation to glycolysis even in an aerobic environment. This phenomenon is known as the Warburg effect. This effect is regulated mainly by polypyrimidine tract-binding protein 1 (PTBP1), which is a splicer of the mRNA for the rate-limiting enzymes of glycolysis, pyruvate kinase muscle 1 and 2 (PKM1 and PKM2). In the present study, we demonstrated that miR-133b reduced PTBP1 expression at translational level and that the expression levels of miR-133b were significantly downregulated in gastric cancer clinical samples and human cell lines, whereas the protein expression level of PTBP1 was upregulated in 80% of the 20 clinical samples of gastric cancer examined. Ectopic expression of miR-133b and knockdown of PTBP1 in gastric cancer cells inhibited cell proliferation through the induction of autophagy by the switching of PKM isoform expression from PKM2-dominant to PKM1-dominant. The growth inhibition was partially canceled by an autophagy inhibitor 3-MA or a reactive oxygen species scavenger N-acetylcysteine. These findings indicated that miR-133b acted as a tumor-suppressor through negative regulation of the Warburg effect in gastric cancer cells.

PKM1 is involved in resistance to anti-cancer drugs.

Resistance to chemotherapy is a crucial problem in the clinical situation. To overcome this issue, many mechanisms of chemoresistance have been elucidated so far. However, this problem still has not been solved completely. In this study, we investigated the mechanism of chemoresistance from the view of cancer metabolism-related genes, especially focusing on the expression profile of pyruvate kinase muscle (PKM) isoforms, which are rate-limiting enzymes in cancer-specific metabolism (Warburg effect). Herein, we showed that PKM1, which promotes oxidative phosphorylation (OXPHOS), was commonly up-regulated in various chemoresistant cells. To clarify the functions of PKM1 in chemoresistance, we investigated effects of PKM1 expression in DLD-1 parental, 5-FU-resistant and oxaliplatin-resistant DLD-1 cells. The overexpression of PKM1 resulted in resistance of the parental cells to 5-FU and oxaliplatin. Moreover, gene-silencing of PKM1 induced apoptosis in these cells including the resistant cells by causing a decrease in the mitochondrial membrane potential. Furthermore, combination therapy using 5-FU or oxaliplatin with siR-PKM1 was also effective against the resistant cells. Our findings should lead to the development of new agents that can cancel the chemoresistance from the view of cancer energy metabolism.