Masato Komatsu

Irinotecan Injures Tight Junction and Causes Bacterial Translocation in Rat

BACKGROUND Tight junctions are an essential component of intestinal epithelial barriers. Claudin-1, occludin, and ZO-1 are the components of tight junction. The purpose of this study was to investigate whether irinotecan induces bacterial translocation in rats, and thus elucidate the relationship between tight junction and bacterial translocation. METHODS Ten rats were divided into two groups: Five were treated with irinotecan and five were not treated with irinotecan, the control group. Irinotecan treated rats were administrated irinotecan 250 mg/kg intraperitoneally on days designated 0 and 1, were then killed at 48 h after treatment, and tissues were collected for analysis. Controls were treated with a saline solution. RESULTS In eighty percent of irinotecan treated rats, bacteria were detected in the mesenteric lymph node or spleen. Large intestinal resistance of the rats was decreased. On the contrary, small intestinal resistance increased. Claudin-1 protein expression of both the small and large intestine decreased (P < 0.05), occludin protein expression of the small intestine decreased (P < 0.05), and occludin protein expression of the large intestine had decreasing tendency (P = 0.07) in irinotecan treated rats. In irinotecan treated rats, claudin-1 mRNA of the small intestine decreased (P < 0.05), claudin-1 mRNA of large intestine had a tendency to decrease (P = 0.05), occludin mRNA of both small and large intestine decreased (P < 0.05). CONCLUSIONS Irinotecan injures claudin-1 and occludin. It causes disorders in the intestinal epithelial barrier and induces bacterial translocation.

Molecular features of triple negative breast cancer cells by genome-wide gene expression profiling analysis

Triple negative breast cancer (TNBC) has a poor outcome due to the lack of beneficial therapeutic targets. To clarify the molecular mechanisms involved in the carcinogenesis of TNBC and to identify target molecules for novel anticancer drugs, we analyzed the gene expression profiles of 30 TNBCs as well as 13 normal epithelial ductal cells that were purified by laser-microbeam microdissection. We identified 301 and 321 transcripts that were significantly upregulated and downregulated in TNBC, respectively. In particular, gene expression profile analyses of normal human vital organs allowed us to identify 104 cancer-specific genes, including those involved in breast carcinogenesis such as NEK2, PBK and MELK. Moreover, gene annotation enrichment analysis revealed prominent gene subsets involved in the cell cycle, especially mitosis. Therefore, we focused on cell cycle regulators, asp (abnormal spindle) homolog, microcephaly-associated (Drosophila) (ASPM) and centromere protein K (CENPK) as novel therapeutic targets for TNBC. Small-interfering RNA-mediated knockdown of their expression significantly attenuated TNBC cell viability due to G1 and G2/M cell cycle arrest. Our data will provide a better understanding of the carcinogenesis of TNBC and could contribute to the development of molecular targets as a treatment for TNBC patients.

Targeting BIG3–PHB2 interaction to overcome tamoxifen resistance in breast cancer cells

The acquisition of endocrine resistance is a common obstacle in endocrine therapy of patients with oestrogen receptor-α (ERα)-positive breast tumours. We previously demonstrated that the BIG3–PHB2 complex has a crucial role in the modulation of oestrogen/ERα signalling in breast cancer cells. Here we report a cell-permeable peptide inhibitor, called ERAP, that regulates multiple ERα-signalling pathways associated with tamoxifen resistance in breast cancer cells by inhibiting the interaction between BIG3 and PHB2. Intrinsic PHB2 released from BIG3 by ERAP directly binds to both nuclear- and membrane-associated ERα, which leads to the inhibition of multiple ERα-signalling pathways, including genomic and non-genomic ERα activation and ERα phosphorylation, and the growth of ERα-positive breast cancer cells both in vitro and in vivo. More importantly, ERAP treatment suppresses tamoxifen resistance and enhances tamoxifen responsiveness in ERα-positive breast cancer cells. These findings suggest inhibiting the interaction between BIG3 and PHB2 may be a new therapeutic strategy for the treatment of luminal-type breast cancer.

Xanthohumol suppresses oestrogen-signalling in breast cancer through the inhibition of BIG3-PHB2 interactions.

Xanthohumol (XN) is a natural anticancer compound that inhibits the proliferation of oestrogen receptor-α (ERα)-positive breast cancer cells. However, the precise mechanism of the antitumour effects of XN on oestrogen (E2)-dependent cell growth, and especially its direct target molecule(s), remain(s) largely unknown. Here, we focus on whether XN directly binds to the tumour suppressor protein prohibitin 2 (PHB2), forming a novel natural antitumour compound targeting the BIG3-PHB2 complex and acting as a pivotal modulator of E2/ERα signalling in breast cancer cells. XN treatment effectively prevented the BIG3-PHB2 interaction, thereby releasing PHB2 to directly bind to both nuclear- and cytoplasmic ERα. This event led to the complete suppression of the E2-signalling pathways and ERα-positive breast cancer cell growth both in vitro and in vivo, but did not suppress the growth of normal mammary epithelial cells. Our findings suggest that XN may be a promising natural compound to suppress the growth of luminal-type breast cancer.

Indoleamine 2,3-dioxygenase Affects the Aggressiveness of Intraductal Papillary Mucinous Neoplasms Through Foxp3(+)CD4(+)CD25(+) T Cells in Peripheral Blood

Objective Intraductal papillary mucinous neoplasms (IPMNs) have a high malignant potential. We previously reported that peripheral Foxp3+CD4+CD25+ T-cell (Foxp3+ Treg) populations significantly increase with IPMN pathological aggressiveness. Dendritic cell-mediated induction of active Tregs from naive CD4+ T cells requires indoleamine 2,3-dioxygenase (IDO). Here, we evaluated whether an IDO–Foxp3+ Treg interaction plays a role in IPMN pathological aggressiveness. Methods We evaluated peripheral blood samples and resected specimens from 12 patients with IPMN. We analyzed Foxp3+CD4+CD25+ T cells in peripheral blood by fluorescence-activated cell sorting, evaluated the resected specimens by anti-IDO antibody staining, and compared them with the patients’ clinicopathological factors. Results The pathological aggressiveness of IPMN was significantly associated with the number of peripheral Foxp3+ Tregs (P < 0.05) and IDO-positive cells per high-power field (HPF) (P < 0.01). There was a significant correlation between the numbers of peripheral Foxp3+ Tregs and IDO-positive cells/HPF (r = 0.625, P < 0.01). Patients with 7 or more IDO-positive cells/HPF had a significantly higher recurrence rate than those with less than 7 IDO-positive cells/HPF (P < 0.01, log-rank test). Conclusions Peripheral Foxp3+ Tregs accurately reflect the aggressiveness of IPMNs. An increase in Foxp3+ Tregs can be induced by local IDO-positive cells in IPMN.

BIG3 Inhibits the Estrogen-Dependent Nuclear Translocation of PHB2 via Multiple Karyopherin-Alpha Proteins in Breast Cancer Cells

We recently reported that brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) binds Prohibitin 2 (PHB2) in cytoplasm, thereby causing a loss of function of the PHB2 tumor suppressor in the nuclei of breast cancer cells. However, little is known regarding the mechanism by which BIG3 inhibits the nuclear translocation of PHB2 into breast cancer cells. Here, we report that BIG3 blocks the estrogen (E2)-dependent nuclear import of PHB2 via the karyopherin alpha (KPNA) family in breast cancer cells. We found that overexpressed PHB2 interacted with KPNA1, KPNA5, and KPNA6, thereby leading to the E2-dependent translocation of PHB2 into the nuclei of breast cancer cells. More importantly, knockdown of each endogenous KPNA by siRNA caused a significant inhibition of E2-dependent translocation of PHB2 in BIG3-depleted breast cancer cells, thereby enhancing activation of estrogen receptor alpha (ERα). These data indicated that BIG3 may block the KPNAs (KPNA1, KPNA5, and KPNA6) binding region(s) of PHB2, thereby leading to inhibition of KPNAs-mediated PHB2 nuclear translocation in the presence of E2 in breast cancer cells. Understanding this regulation of PHB2 nuclear import may provide therapeutic strategies for controlling E2/ERα signals in breast cancer cells.

Therapeutic advances in BIG3-PHB2 inhibition targeting the crosstalk between estrogen and growth factors in breast cancer

Our previous studies demonstrated that specific inhibition of the BIG3‐PHB2 complex, which is a critical modulator in estrogen (E2) signaling, using ERAP, a dominant negative peptide inhibitor, leads to suppression of E2‐dependent estrogen receptor (ER) alpha activation through the reactivation of the tumor suppressive activity of PHB2. Here, we report that ERAP has significant suppressive effects against synergistic activation caused by the crosstalk between E2 and growth factors associated with intrinsic or acquired resistance to anti‐estrogen tamoxifen in breast cancer cells. Intrinsic PHB2 released from BIG3 by ERAP effectively disrupted each interaction of membrane‐associated ERα and insulin‐like growth factor 1 receptor beta (IGF‐1Rβ), EGFR, PI3K or human epidermal growth factor 2 (HER2) in the presence of E2 and the growth factors IGF or EGF, followed by inhibited the activation of IGF‐1Rβ, EGFR or HER2, and reduced Akt, MAPK and ERα phosphorylation levels, resulting in significant suppression of proliferation of ERα‐positive breast cancer cells in vitro and in vivo. More importantly, combined treatment with ERAP and tamoxifen led to a synergistic suppression of signaling that was activated by crosstalk between E2 and growth factors or HER2 amplification. Taken together, our findings suggest that the specific inhibition of BIG3‐PHB2 is a novel potential therapeutic approach for the treatment of tamoxifen‐resistant breast cancers activated by the crosstalk between E2 and growth factor signaling, especially in premenopausal women.

Early growth response 4 is involved in cell proliferation of small cell lung cancer through transcriptional activation of its downstream genes.

Small cell lung cancer (SCLC) is aggressive, with rapid growth and frequent bone metastasis; however, its detailed molecular mechanism remains poorly understood. Here, we report the critical role of early growth factor 4 (EGR4), a DNA-binding, zinc-finger transcription factor, in cell proliferation of SCLC. EGR4 overexpression in HEK293T cells conferred significant upregulation of specific splice variants of the parathyroid hormone-related protein (PTHrP) gene, resulting in enhancement of the secretion of PTHrP protein, a known mediator of osteolytic bone metastasis. More importantly, depletion of EGR4 expression by siRNA significantly suppressed growth of the SCLC cell lines, SBC-5, SBC-3 and NCI-H1048. On the other hand, introduction of EGR4 into NIH3T3 cells significantly enhanced cell growth. We identified four EGR4 target genes, SAMD5, RAB15, SYNPO and DLX5, which were the most significantly downregulated genes upon depletion of EGR4 expression in all of the SCLC cells examined, and demonstrated the direct recruitment of EGR4 to their promoters by ChIP and luciferase reporter analysis. Notably, knockdown of the expression of these genes by siRNA remarkably suppressed the growth of all the SCLC cells. Taken together, our findings suggest that EGR4 likely regulates the bone metastasis and proliferation of SCLC cells via transcriptional regulation of several target genes, and may therefore be a promising target for the development of anticancer drugs for SCLC patients.

Involvement of B3GALNT2 overexpression in the cell growth of breast cancer

A number of glycosyltransferases have been identified and biologically characterized in cancer cells, yet their exact pathophysiological functions are largely unknown. Here, we report the critical role of β1,3-N-acetylgalactosaminyltransferase II (B3GALNT2), which transfers N-acetylgalactosamine (GalNAc) in a β1,3 linkage to N-acetylglucosamine, in the growth of breast cancer cells. Comprehensive transcriptomics, quantitative PCR and northern blot analyses indicated this molecule to be exclusively upregulated in the majority of breast cancers. Knockdown of B3GALNT2 expression by small interfering RNA attenuated cell growth and induced apoptosis in breast cancer cells. Overexpression of B3GALNT2 in HEK293T cells prompted secretion of the gene product into the culture medium, suggesting that B3GALNT2 is potentially a secreted protein. Furthermore, we demonstrated that B3GALNT2 is N-glycosylated on both Asn-116 and Asn-174 and that this modification is necessary for its secretion in breast cancer cells. Our findings suggest that this molecule represents a promising candidate for the development of a novel therapeutic targeting drug and a potential diagnostic tumor marker for patients with breast cancer, especially TNBC.

Stapled BIG3 helical peptide ERAP potentiates anti-tumour activity for breast cancer therapeutics

Estradiol (E2) and the oestrogen receptor-alpha (ERα) signalling pathway play pivotal roles in the proliferative activity of breast cancer cells. Recent findings show that the brefeldin A-inhibited guanine nucleotide-exchange protein 3-prohibitin 2 (BIG3-PHB2) complex plays a crucial role in E2/ERα signalling modulation in breast cancer cells. Moreover, specific inhibition of the BIG3-PHB2 interaction using the ERα activity-regulator synthetic peptide (ERAP: 165–177 amino acids), derived from α-helical BIG3 sequence, resulted in a significant anti-tumour effect. However, the duration of this effect was very short for viable clinical application. We developed the chemically modified ERAP using stapling methods (stapledERAP) to improve the duration of its antitumour effects. The stapledERAP specifically inhibited the BIG3-PHB2 interaction and exhibited long-lasting suppressive activity. Its intracellular localization without the membrane-permeable polyarginine sequence was possible via the formation of a stable α-helix structure by stapling. Tumour bearing-mice treated daily or weekly with stapledERAP effectively prevented the BIG3-PHB2 interaction, leading to complete regression of E2-dependent tumours in vivo. Most importantly, combination of stapledERAP with tamoxifen, fulvestrant, and everolimus caused synergistic inhibitory effects on growth of breast cancer cells. Our findings suggested that the stapled ERAP may be a promising anti-tumour drug to suppress luminal-type breast cancer growth.