The net acid extruders NHE1, NBCn1 and MCT4 promote mammary tumor growth through distinct but overlapping mechanisms

High metabolic and proliferative rates in cancer cells lead to production of large amounts of H⁺ and CO₂, and as a result, net acid extruding transporters are essential for the function and survival of cancer cells. We assessed protein expression of the Na⁺/H⁺ exchanger NHE1, the Na⁺‐ cotransporter NBCn1, and the lactate‐H⁺ cotransporters MCT1 and ?4 by immunohistochemical analysis of a large cohort of breast cancer samples. We found robust expression of these transporters in 20, 10, 4 and 11% of samples, respectively. NHE1 and NBCn1 expression both correlated positively with progesterone receptor status, NHE1 correlated negatively and NBCn1 positively with HER2 status, whereas MCT4 expression correlated with lymph node status. Stable shRNA‐mediated knockdown (KD) of either NHE1 or NBCn1 in the MDA‐MB‐231 triple‐negative breast cancer (TNBC) cell line significantly reduced steady‐state intracellular pH (pH_i) and capacity for pH_i recovery after an acid load. Importantly, KD of any of the three transporters reduced in vivo primary tumor growth of MDA‐MB‐231 xenografts. However, whereas KD of NBCn1 or MCT4 increased tumor‐free survival and decreased in vitro proliferation rate and colony growth in soft agar, KD of NHE1 did not have these effects. Moreover, only MCT4 KD reduced Akt kinase activity, PARP and CD147 expression and cell motility. This work reveals that different types of net acid extruding transporters, NHE1, NBCn1 and MCT4, are frequently expressed in patient mammary tumor tissue and demonstrates for the first time that they promote growth of TNBC human mammary tumors in vivo via distinct but overlapping mechanisms.     

The sodium channel β1 subunit mediates outgrowth of neurite‐like processes on breast cancer cells and promotes tumour growth and metastasis

Voltage‐gated Na⁺ channels (VGSCs) are heteromeric proteins composed of pore‐forming α subunits and smaller β subunits. The β subunits are multifunctional channel modulators and are members of the immunoglobulin superfamily of cell adhesion molecules (CAMs). β1, encoded by SCN1B, is best characterized in the central nervous system (CNS), where it plays a critical role in regulating electrical excitability, neurite outgrowth and migration during development. β1 is also expressed in breast cancer (BCa) cell lines, where it regulates adhesion and migration in vitro. In the present study, we found that SCN1B mRNA/β1 protein were up‐regulated in BCa specimens, compared with normal breast tissue. β1 upregulation substantially increased tumour growth and metastasis in a xenograft model of BCa. β1 over‐expression also increased vascularization and reduced apoptosis in the primary tumours, and β1 over‐expressing tumour cells had an elongate morphology. In vitro, β1 potentiated outgrowth of processes from BCa cells co‐cultured with fibroblasts, via trans‐homophilic adhesion. β1‐mediated process outgrowth in BCa cells required the presence and activity of fyn kinase, and Na⁺ current, thus replicating the mechanism by which β1 regulates neurite outgrowth in CNS neurons. We conclude that when present in breast tumours, β1 enhances pathological growth and cellular dissemination. This study is the first demonstration of a functional role for β1 in tumour growth and metastasis in vivo. We propose that β1 warrants further study as a potential biomarker and targeting β1‐mediated adhesion interactions may have value as a novel anti‐cancer therapy.     

Transport by SLC5A8 with subsequent inhibition of histone deacetylase 1 (HDAC1) and HDAC3 underlies the antitumor activity of 3‐bromopyruvate

BACKGROUND:

3‐Bromopyruvate is an alkylating agent with antitumor activity. It is currently believed that blockade of adenosine triphosphate production from glycolysis and mitochondria is the primary mechanism responsible for this antitumor effect. The current studies uncovered a new and novel mechanism for the antitumor activity of 3‐bromopyruvate.

METHODS:

The transport of 3‐bromopyruvate by sodium‐coupled monocarboxylate transporter SMCT1 (SLC5A8), a tumor suppressor and a sodium (Na⁺)‐coupled, electrogenic transporter for short‐chain monocarboxylates, was studied using a mammalian cell expression and the Xenopus laevis oocyte expression systems. The effect of 3‐bromopyruvate on histone deacetylases (HDACs) was monitored using the lysate of the human breast cancer cell line MCF7 and human recombinant HDAC isoforms as the enzyme sources. Cell viability was monitored by fluorescence‐activated cell‐sorting analysis and colony‐formation assay. The acetylation status of histone H4 was evaluated by Western blot analysis.

RESULTS:

3‐Bromopyruvate is a transportable substrate for SLC5A8, and that transport process is Na⁺‐coupled and electrogenic. MCF7 cells did not express SLC5A8 and were not affected by 3‐bromopyruvate. However, when transfected with SLC5A8 or treated with inhibitors of DNA methylation, these cells underwent apoptosis in the presence of 3‐bromopyruvate. This cell death was associated with the inhibition of HDAC1/HDAC3. Studies with different isoforms of human recombinant HDACs identified HDAC1 and HDAC3 as the targets for 3‐bromopyruvate.

CONCLUSIONS:

3‐Bromopyruvate was transported into cells actively through the tumor suppressor SLC5A8, and the process was energized by an electrochemical Na⁺ gradient. Ectopic expression of the transporter in MCF7 cells led to apoptosis, and the mechanism involved the inhibition of HDAC1/HDAC3. Cancer 2009. © 2009 American Cancer Society.     

Association of breast cancer risk loci with breast cancer survival

The survival of breast cancer patients is largely influenced by tumor characteristics, such as TNM stage, tumor grade and hormone receptor status. However, there is growing evidence that inherited genetic variation might affect the disease prognosis and response to treatment. Several lines of evidence suggest that alleles influencing breast cancer risk might also be associated with breast cancer survival. We examined the associations between 35 breast cancer susceptibility loci and the disease over‐all survival (OS) in 10,255 breast cancer patients from the National Cancer Institute Breast and Prostate Cancer Cohort Consortium (BPC3) of which 1,379 died, including 754 of breast cancer. We also conducted a meta‐analysis of almost 35,000 patients and 5,000 deaths, combining results from BPC3 and the Breast Cancer Association Consortium (BCAC) and performed in silico analyses of SNPs with significant associations. In BPC3, the C allele of LSP1‐rs3817198 was significantly associated with improved OS (HR_per‐allele=0.70; 95% CI: 0.58–0.85; p_trend = 2.84 × 10^?4; HR_{heterozygotes} = 0.71; 95% CI: 0.55–0.92; HR_homozygotes = 0.48; 95% CI: 0.31–0.76; p_2DF = 1.45 × 10^?3). In silico, the C allele of LSP1‐rs3817198 was predicted to increase expression of the tumor suppressor cyclin‐dependent kinase inhibitor 1C (CDKN1C). In the meta‐analysis, TNRC9‐rs3803662 was significantly associated with increased death hazard (HR_META =1.09; 95% CI: 1.04–1.15; p_trend = 6.6 × 10^?4; HR_{heterozygotes} = 0.96 95% CI: 0.90–1.03; HR_homozygotes = 1.21; 95% CI: 1.09–1.35; p_2DF=1.25 × 10^?4). In conclusion, we show that there is little overlap between the breast cancer risk single nucleotide polymorphisms (SNPs) identified so far and the SNPs associated with breast cancer prognosis, with the possible exceptions of LSP1‐rs3817198 and TNRC9‐rs3803662.     

Interleukin‐33/ST2 axis promotes breast cancer growth and metastases by facilitating intratumoral accumulation of immunosuppressive and innate lymphoid cells

The role of IL‐33/ST2 pathway in antitumor immunity is unclear. Using 4T1 breast cancer model we demonstrate time‐dependent increase of endogenous IL‐33 at both the mRNA and protein levels in primary tumors and metastatic lungs during cancer progression. Administration of IL‐33 accelerated tumor growth and development of lung and liver metastases, which was associated with increased intratumoral accumulation of CD11b⁺Gr‐1⁺ TGF‐β1⁺ myeloid‐derived suppressor cells (MDSCs) that expressed IL‐13α1R, IL‐13‐producing Lin^?Sca‐1⁺ST2⁺ innate lymphoid cells (ILCs) and CD4⁺Foxp3⁺ST2⁺IL‐10⁺ Tregs compared to untreated mice. Higher incidence of monocytic vs. granulocytic MDSCs and plasmocytoid vs. conventional dendritic cells (DCs) was present in mammary tumors of IL‐33‐treated mice. Intratumoral NKp46⁺NKG2D⁺ and NKp46⁺FasL⁺ cells were markedly reduced after IL‐33 treatment, while phosphate‐buffered saline‐treated ST2‐deficient mice had increased frequencies of these tumoricidal natural killer (NK) cells compared to untreated wild‐type mice. IL‐33 promoted intratumoral cell proliferation and neovascularization, which was attenuated in the absence of ST2. Tumor‐bearing mice given IL‐33 had increased percentages of splenic MDSCs, Lin^?Sca‐1⁺ ILCs, IL‐10‐expressing CD11c⁺ DCs and alternatively activated M2 macrophages and higher circulating levels of IL‐10 and IL‐13. A significantly reduced NK cell, but not CD8⁺ T‐cell cytotoxicity in IL‐33‐treated mice was observed and the mammary tumor progression was not affected when CD8⁺ T cells were in vivo depleted. We show a previously unrecognized role for IL‐33 in promoting breast cancer progression through increased intratumoral accumulation of immunosuppressive cells and by diminishing innate antitumor immunity. Therefore, IL‐33 may be considered as an important mediator in the regulation of breast cancer progression.     

Hypoxia promotes the phenotypic change of aldehyde dehydrogenase activity of breast cancer stem cells

Stable breast cancer cell (BCC) lines are valuable tools for the identification of breast cancer stem cell (BCSC) phenotypes that develop in response to several stimuli as well as for studying the basic mechanisms associated with the initiation and maintenance of BCSCs. However, the characteristics of individual, BCC‐derived BCSCs varies and these cells show distinct phenotypes depending on the different BCSC markers used for their isolation. Aldehyde dehydrogenase (ALDH) activity is just such a recognized biomarker of BCSCs with a CD44⁺/CD24^? phenotype. We isolated BCSCs with high ALDH activity (CD44⁺/CD24^?/Aldefluor^pos) from a primary culture of human breast cancer tissue and observed that the cells had stem cell properties compared to BCSCs with no ALDH activity (CD44⁺/CD24^?/Aldefluor^neg). Moreover, we found Aldefluor^pos BCSCs had a greater hypoxic response and subsequent induction of HIF‐1α expression compared to the Aldefluor^neg BCSCs. We also found that knocking down HIF‐1α, but not HIF‐2α, in Aldefluor^pos BCSCs led to a significant reduction of the stem cell properties through a decrease in the mRNA levels of genes associated with the epithelial‐mesenchymal transition. Indeed, HIF‐1α overexpression in Aldefluor^neg BCSCs led to Slug and Snail mRNA increase and the associated repression of E‐cadherin and increase in Vimentin. Of note, prolonged hypoxic stimulation promoted the phenotypic changes of Aldefluor^neg BCSCs including ALDH activity, tumorigenesis and metastasis, suggesting that hypoxia in the tumor environment may influence BCSC fate and breast cancer clinical outcomes.     

Comparison of tumor‐infiltrating lymphocytes between primary and metastatic tumors in breast cancer patients

The presence of tumor‐infiltrating lymphocytes (TILs) is associated with favorable long‐term outcome in breast cancer. However, little is known about changes in TILs during metastatic progression. To confirm our hypothesis that malignant tumors escape from the host immune system during metastasis, we evaluated the percentage of TILs in paired samples of primary and metastatic breast tumors. We retrospectively identified 25 patients with human epidermal growth factor receptor‐2 (HER2⁺, n = 14) and triple negative (TN, n = 11) early breast cancer diagnosed between 1990 and 2009 at Tokai University Hospital (Isehara, Japan) and who subsequently experienced regional or distant recurrence confirmed by tumor biopsy/resection. Hematoxylin–eosin‐stained slides of these paired samples were evaluated for stromal TILs. Immunohistochemical staining was carried out using primary antibodies against CD4, CD8, Foxp3, programmed cell death ligand 1 (PD‐L1), PD‐L2, and HLA class I for characterizing the TILs and breast tumors. The percentage of TILs in the primary tumors was significantly higher (average 34.6%) than that in metastatic tumors (average 15.7%) (paired t‐test, P = 0.004) and that of CD8⁺ and CD4⁺ T cells significantly decreased from primary to metastatic tumors (paired t‐test, P = 0.008 and P = 0.026, respectively). The PD‐L1, PD‐L2, and HLA class I antibody expression changed from positive to negative and vice versa from the primary to the metastatic tumors. Tumors at first metastatic recurrence in HER2⁺ and TN breast cancers have a lower percentage of TILs and CD8⁺ and CD4⁺ T cells compared to primary tumors, which indicates that immune escape plays a role in tumor progression.     

Expression of estrogen,progesterone and epidermal growth factor receptors in primary and metastatic breast cancer

The prognostic value of epidermal growth factor receptor (EGFR) expression and its biological role in estrogen receptor-positive (ER⁺) and ER-negative (ER^?) primary breast cancer is controversial. In this study, distributions of ER, progesterone receptor and EGFR have been established using immunohisto-chemistry in both primary breast tumors and their matched axillary lymph node metastases of 26 patients or their matched distant metastases of 2 patients. In addition, 5 patients with bilateral breast cancer were studied. ER⁺ tumor cells were detected in 22 (69%) and EGFR⁺ tumor cells were detected in 11 (34%) primary breast carcinomas. Expression of ER and EGFR was inverse regarding the individual tumor cells in both primary tumors and metastases. Relationship of EGFR expression with poorly differentiated and large breast tumors was observed. Furthermore, primary tumors with a predominant lobular component were ER⁺ and, with one exception, EGFR^?. Invasive ductal carcinomas were more frequently EGFR⁺. No apparent differences in receptor expression were observed between primary tumors and lymph node metastases or chro-nously or metachronously occurring bilateral breast cancers. Only one ER⁺ primary tumor showed a switch to EGFR expression in the involved lymph node. Our study shows that a shift in receptor phenotype between primary tumors and lymph node metastases is a rare event and, thus, additional analyses of involved lymph nodes will not likely serve as a better predictor for response to anti-estrogen therapy. We conclude that expression of EGFR is not a prerequisite for development of metastases. © 1995 Wiley-Liss, Inc.     

Immunological evaluation of peptide vaccination for cancer patients with the HLA ‐A11+ or ‐A33+ allele

The HLA‐A11 or ‐A33 allele is found in approximately 18% or 10% of the Asian population, respectively, but each of which is a minor allele worldwide, and therefore no clinical trials were previously conducted. To develop a therapeutic peptide vaccine for each of them, we investigated immunological responses of advanced cancer patients with the HLA‐A11⁺/A11⁺ (n = 18) or ‐A33⁺/A33⁺ (n = 13) allele to personalized peptide vaccine (PPV) regimens. The primary sites of HLA‐A11+/A11+ or ‐A33+/A33+ patients were the colon (n = 4 or 2), stomach (2 or 3), breast (3 or 2), lung and pancreas (2 or 2), and so on. For PPV, a maximum of four peptides were selected from nine different peptides capable of binding to HLA‐A11 and ‐A33 molecules based on the pre‐existing peptide‐specific IgG responses. There were no severe adverse events related to PPV. At the end of the first cycle, peptide‐specific CTL responses were augmented in 4/12 or 2/9 of HLA‐A11⁺/A11⁺ or ‐A33⁺/A33⁺ patients, while peptide‐specific IgG responses were augmented in 6/14 or 4/10 patients, respectively. Clinical responses consisted of four stable diseases and 14 progressive diseases in HLA‐A11⁺/A11⁺patients, versus seven and six in ‐A33⁺/A33⁺patients, respectively. Further clinical study of PPV could be recommended because of the safety and positive immunological responses.     

Diosgenin targets Akt‐mediated prosurvival signaling in human breast cancer cells

In recent years, Akt signaling has gained recognition for its functional role in more aggressive, therapy‐resistant malignancies. As it is frequently constitutively active in cancer cells, several drugs are being investigated for their ability to inhibit Akt signaling. The purpose of this study is to determine effect of diosgenin (fenugreek), a dietary compound on Akt signaling and its downstream targets on estrogen receptor positive (ER⁺) and estrogen receptor negative (ER^?) breast cancer (BCa) cells. Diosgenin inhibits pAkt expression and Akt kinase activity without affecting PI3 kinase levels, resulting in the inhibition of its downstream targets, NF‐κB, Bcl‐2, survivin and XIAP. The Raf/MEK/ERK pathway, another functional downstream target of Akt, was inhibited by diosgenin in ER⁺ but not in ER^? BCa cells. Additionally, we found that diosgenin caused G1 cell cycle arrest by downregulating cyclin D1, cdk‐2 and cdk‐4 expression in both ER⁺ and ER^? BCa cells resulting in the inhibition of cell proliferation and induction of apoptosis. Interestingly, no significant toxicity was seen in the normal breast epithelial cells (MCF‐10A) following treatment with diosgenin. Additionally, in vivo tumor studies indicate diosgenin significantly inhibits tumor growth in both MCF‐7 and MDA‐231 xenografts in nude mice. Thus, these results suggest that diosgenin might prove to be a potential chemotherapeutic agent for the treatment of BCa. © 2009 UICC     

The risk of a second primary lung cancer after a first invasive breast cancer according to estrogen receptor status

Purpose

Lung cancers account for 5?% of second primary cancers after breast cancer. The low overall 5-year relative survival rate of lung cancer makes it a particularly concerning new malignancy for breast cancer survivors. It is unknown whether second lung cancer risk varies by estrogen receptor (ER) expression of the first breast cancer.

Methods

We evaluated second primary lung cancer risks using standardized incidence ratios (SIRs) (95?% confidence intervals (CIs)) among 222,148 one-year breast cancer survivors in the NCI-SEER Program registry database (1992–2008). Relative risks (RRs) and 95?% CIs for lung cancer following ER^? compared with ER⁺ breast cancer were estimated using Poisson regression, adjusted for age, year, and stage of breast cancer diagnosis, attained age, latency, and radiotherapy. We also examined the reciprocal association of second ER^? and ER⁺ breast cancers among 28,107 1-year lung cancer survivors.

Results

There were 418 and 1,444 second lung cancers diagnosed following 50,781 ER^? and 171,367 ER⁺ breast cancers. Second lung cancer rates were significantly elevated after ER^? (SIR?=?1.20 (1.09–1.33)), but not ER⁺ (SIR?=?0.96 (0.91–1.01)) breast cancer. The adjusted RR for a second lung cancer following ER^? compared with ER⁺ breast cancer was 1.22 (1.10–1.37). The reciprocal adjusted RR for a second ER^? compared with ER⁺ breast cancer following lung cancer was 1.29 (0.98–1.70).

Conclusion

The parallel increase for a second lung cancer following an ER^? first breast cancer and for a second ER^? breast cancer after a first lung cancer suggests that there may be shared etiologic factors for these cancers. Further evaluation of lung cancer risk after ER^? breast cancer may identify women at high risk for this fatal malignancy.     

Expression of the SART‐1 tumor rejection antigen in breast cancer

We investigated in breast cancers the expression of the SART‐1 gene encoding tumor rejection antigens. SART‐1 mRNA was expressed in all of the samples tested. The SART‐1₈₀₀ antigen was detectable in 20 of 50 (40%) breast cancer tissues and all breast cancer cell lines tested, but not in normal breast tissues. The SART‐1₈₀₀⁺ breast cancer cells transfected with HLA‐A2601 or HLA‐A2402 cDNA were recognized by the HLA‐A26‐restricted and SART‐1‐specific cytotoxic T lymphocytes (CTLs) or the HLA‐A24‐restricted and SART‐1‐specific CTLs, respectively. Among the 20 SART‐1₈₀₀⁺ tumors, 9 or 8 tumors expressed estrogen receptor or progesterone receptor, respectively. Therefore, the patients with HLA‐A26 or ‐A24 haplotype might be appropriate candidates for specific immunotherapy with the SART‐1 peptides independently or in combination with hormone therapy. Int. J. Cancer 80:64–67, 1999. © 1999 Wiley‐Liss, Inc.     

Silencing IDO in dendritic cells: A novel approach to enhance cancer immunotherapy in a murine breast cancer model

Cancer immunotherapeutic agents (vaccines) in the form of antigen‐loaded dendritic cells (DCs) reached an important milestone with the recent approval of Provenge, the first DC vaccine for treatment of prostate cancer. Although this heralds a new era of tumor immunotherapy, it also highlights the compelling need to optimize such DC‐based therapies as they are increasingly tested and used to treat human patients. In this study we sought to augment and enhance the antitumor activity of a DC‐based vaccine using siRNA to silence expression of immunosuppressive enzyme indoleamine 2,3‐dioxygenase (IDO) in DCs. We report here that DCs loaded with tumor antigens, but with siRNA‐silenced IDO expression, were introduced into 4T1 breast tumor‐bearing mice, the treatment: (i) lengthened the time required for tumor onset, (ii) decreased tumor size compared to tumors grown for equal lengths of time in mice treated with antigen‐loaded DCs without IDO silencing and (iii) reduced CD4⁺ and CD8⁺ T cell apoptosis. Furthermore, immunization with IDO‐silenced DCs enhanced tumor antigen‐specific T cell proliferation and CTL activity, and decreased numbers of CD4⁺CD25⁺Foxp3⁺ T_reg. This study provides evidence to support silencing of immunosuppressive genes (IDO) as an effective strategy to enhance the efficacy of DC‐based cancer immunotherapeutic.     

MicroRNA‐93 targets WASF3 and functions as a metastasis suppressor in breast cancer

Cancer cells with cancer stem cell (CSC) properties initiate both primary tumor formation and metastases at distant sites. Acquisition of CSC properties is highly associated with epigenetic alterations, including those mediated by microRNAs (miRNAs). We have previously established the breast cancer patient‐derived tumor xenograft (PDX) mouse model in which CSC marker CD44⁺ cancer cells formed spontaneous microscopic metastases in the liver. In this PDX mouse, we found that the expression levels of 3 miRNAs (miR‐25, miR‐93, and miR‐106b) in the miR‐106b‐25 cluster were much lower in the CD44⁺ human cancer cells metastasized to the liver than those at the primary site. Constitutive overexpression of miR‐93 suppressed invasive ability and 3D‐organoid formation capacity of breast cancer cells in vitro and significantly suppressed their metastatic ability to the liver in vivo. Wiskott‐Aldrich syndrome protein family member 3 (WASF3), a regulator of both cytoskeleton remodeling and CSC properties, was identified as a functional target of miR‐93: overexpression of miR‐93 reduced the protein level of WASF3 in breast cancer cells and WASF3 rescued the miR‐93‐mediated suppression of breast cancer cell invasion. These findings suggest that miR‐93 functions as a metastasis suppressor by suppressing both invasion ability and CSC properties in breast cancers.     

Voltage‐gated sodium channel Nav1.7 promotes gastric cancer progression through MACC1‐mediated upregulation of NHE1

Voltage‐gated sodium channels (VGSCs), which are aberrantly expressed in several human cancers, affect cancer cell behavior; however, their role in gastric cancer (GC) and the link between these channels and tumorigenic signaling remain unclear. The aims of this study were to determine the clinicopathological significance and role of the VGSC Na_v1.7 in GC progression and to investigate the associated mechanisms. Here, we report that the SCN9A gene encoding Na_v1.7 was the most abundantly expressed VGSC subtype in GC tissue samples and two GC cell lines (BGC‐823 and MKN‐28 cells). SCN9A expression levels were also frequently found to be elevated in GC samples compared to nonmalignant tissues by real‐time PCR. In the 319 GC specimens evaluated by immunohistochemistry, Na_v1.7 expression was correlated with prognosis, and transporter Na⁺/H⁺ exchanger‐1 (NHE1) and oncoprotein metastasis‐associated in colon cancer‐1 (MACC1) expression. Na_v1.7 suppression resulted in reduced voltage‐gated sodium currents, decreased NHE1 expression, increased extracellular pH and decreased intracellular pH, and ultimately, reduced invasion and proliferation rates of GC cells and growth of GC xenografts in nude mice. Na_v1.7 inhibition led to reduced MACC1 expression, while MACC1 inhibition resulted in reduced NHE1 expression in vitro and in vivo. Mechanistically, the suppression of Na_v1.7 decreased NF‐κB p65 nuclear translocation via p38 activation, thus reducing MACC1 expression. Downregulation of MACC1 decreased c‐Jun phosphorylation and subsequently reduced NHE1 expression, whereas the addition of hepatocyte growth factor (HGF), a c‐Met physiological ligand, reversed the effect. These results indicate that Na_v1.7 promotes GC progression through MACC1‐mediated upregulation of NHE1. Therefore, Na_v1.7 is a potential prognostic marker and/or therapeutic target for GC.     

Nigericin enhances mafosfamide cytotoxicity at low extracellular pH

Summary The cytotoxicity of many alkylating anticancer drugs is increased at reduced intracellular pH (pH_i). The therapeutic index of such agents could therefore be improved by lowering pH_i in the target cells prior to their application. We have previously demonstrated that the formation of lactic acid can be selectively enhanced in malignant tissues via glucose-mediated stimulation of tumor cell glycolysis. However, the resulting reduction in pH_i is partly compensated by the extrusion of H⁺ equivalents into the extracellular space, with pH_i remaining closer to the physiological value than extracellular pH (pH_e). For full exploitation of the proton-mediated increase in the cytotoxicity of alkylating agents, pH_i should therefore be equilibrated with pH_e in lactic acid-producing cells. In the present study we investigated the question as to whether nigericin, an H⁺/K⁺ antiporter enabling the entry into cells of H⁺ ions at low pH_e, can be used to enhance the cytotoxic effect of mafosfamide (MAFO; a precursor of activated cyclophosphamide) on cultured M1R rat mammary carcinoma cells. At pH_e 7.4, the cytotoxic effect of combined treatment with MAFO and nigericin was not superior to treatment with MAFO alone. At acidic pH_e, however, MAFO cytotoxicity was potentiated by nigericin as indicated by the colony-forming capacity of M1R cells. For example, at pH_e 6.2 (corresponding to the approximate mean aggregated pH in actively glycolyzing tumors), the colonyforming fraction of cells treated with a combination of MAFO and nigericin was 3×10^–5 that of controls, as compared with a value of 5×10^–2 found for cells exposed to MAFO alone. These results suggest that agents counteracting cellular mechanisms that control pH_i may be candidate compounds for investigations aimed at the enhancement of alkylating drug cytotoxicity following glucosemediated pH reduction in malignant tumors in vivo.This work was supported by grants from the Dr. Mildred Scheel-Stiftung für Krebsforschung and by the Federal Ministry for Research and Technology (0318849 A)     

Identification of NY‐BR‐1‐specific CD4+ T cell epitopes using HLA‐transgenic mice

Breast cancer represents the second most common cancer type worldwide and has remained the leading cause of cancer‐related deaths among women. The differentiation antigen NY‐BR‐1 appears overexpressed in invasive mammary carcinomas compared to healthy breast tissue, thus representing a promising target antigen for T cell based tumor immunotherapy approaches. Since efficient immune attack of tumors depends on the activity of tumor antigen‐specific CD4⁺ effector T cells, NY‐BR‐1 was screened for the presence of HLA‐restricted CD4⁺ T cell epitopes that could be included in immunological treatment approaches. Upon NY‐BR‐1‐specific DNA immunization of HLA‐transgenic mice and functional ex vivo analysis, a panel of NY‐BR‐1‐derived library peptides was determined that specifically stimulated IFNγ secretion among splenocytes of immunized mice. Following in silico analyses, four candidate epitopes were determined which were successfully used for peptide immunization to establish NY‐BR‐1‐specific, HLA‐DRB1*0301– or HLA‐DRB1*0401‐restricted CD4⁺ T cell lines from splenocytes of peptide immunized HLA‐transgenic mice. Notably, all four CD4⁺ T cell lines recognized human HLA‐DR‐matched dendritic cells (DC) pulsed with lysates of NY‐BR‐1 expressing human tumor cells, demonstrating natural processing of these epitopes also within the human system. Finally, CD4⁺ T cells specific for all four CD4⁺ T cell epitopes were detectable among PBMC of breast cancer patients, showing that CD4⁺ T cell responses against the new epitopes are not deleted nor inactivated by self‐tolerance mechanisms. Our results present the first NY‐BR‐1‐specific HLA‐DRB1*0301– and HLA‐DRB1*0401‐restricted T cell epitopes that could be exploited for therapeutic intervention against breast cancer.     

Cleavage of galectin‐3 by matrix metalloproteases induces angiogenesis in breast cancer

Galectin‐3 cleavage is related to progression of human breast and prostate cancer and is partly responsible for tumor growth, angiogenesis and apoptosis resistance in mouse models. A functional polymorphism in galectin‐3 gene, determining its susceptibility to cleavage by matrix metalloproteinases (MMPs)‐2/‐9 is related to racial disparity in breast cancer incidence in Asian and Caucasian women. The purpose of our study is to evaluate (i) if cleavage of galectin‐3 could be related to angiogenesis during the progression of human breast cancer, (ii) the role of cleaved galectin‐3 in induction of angiogenesis and (iii) determination of the galectin‐3 domain responsible for induction of angiogenic response. Galectin‐3 null breast cancer cells BT‐459 were transfected with either cleavable full‐length galectin‐3 or its fragmented peptides. Chemotaxis, chemoinvasion, heterotypic aggregation, epithelial‐endothelial cell interactions and angiogenesis were compared to noncleavable galectin‐3. BT‐549‐H⁶⁴ cells harboring cleavable galectin‐3 exhibited increased chemotaxis, invasion and interactions with endothelial cells resulting in angiogenesis and 3D morphogenesis compared to BT‐549‐P⁶⁴ cells harboring noncleavable galectin‐3. BT‐549‐H⁶⁴ cells induced increased migration and phosphorylation of focal adhesion kinase in migrating endothelial cells. Endothelial cells cocultured with BT‐549 cells transfected with galectin‐3 peptides indicate that amino acids 1–62 and 33–250 stimulate migration and morphogenesis of endothelial cells. Immunohistochemical analysis of blood vessel density and galectin‐3 cleavage in a breast cancer progression tissue array support the in vitro findings. We conclude that the cleavage of the N terminus of galectin‐3 followed by its release in the tumor microenvironment in part leads to breast cancer angiogenesis and progression.     

Endogenous conversion of ω‐6 to ω‐3 polyunsaturated fatty acids in fat‐1 mice attenuated intestinal polyposis by either inhibiting COX‐2/β‐catenin signaling or activating 15‐PGDH/IL‐18

Omega‐3 polyunsaturated fatty acids (ω‐3PUFAs) have inhibitory effects in various preclinical cancer models, but their effects in intestinal polyposis have never been examined. As attempts have been made to use nutritional intervention to counteract colon cancer development, in this study we evaluated the effects of ω‐3 PUFAs on intestinal polyposis in the Apc^Min/+ mouse model. The experimental groups included wild‐type C56BL/6 mice, Apc^Min/+ mice, fat‐1 transgenic mice expressing an n‐3 desaturase to enable ω‐3 PUFA synthesis, and Apc^Min/+ × fat‐1 double‐transgenic mice; all mice were 20 weeks of age. Small intestines were collected for gross and pathologic evaluation, including assessment of polyp number and size, followed by immunohistochemical staining and Western blotting. After administration of various concentrations of ω‐3 PUFAs, PUFA levels were measured in small intestine tissue by GC/MS/MS analysis to compare with PUFA synthesis of between C57BL6 and fat‐1mice. As a result, ω‐3 PUFAs significantly attenuated Apc mutation–induced intestinal polyposis accompanied with significant inhibition of Wnt/β‐catenin signaling, COX‐2 and PGE_2, but induced significant levels of 15‐PGDH. In addition, significant induction of the inflammasome‐related substrates as IL‐1β and IL‐18 and activation of caspase‐1 was observed in Apc^Min/+ × fat‐1 mice. Administration of at least 3 g/60 kg ω‐3 PUFAs was equivalent to ω‐3 PUFAs produced in fat‐1 mice and resulted in significant increase in the expression of IL‐1β, caspase‐3 and IL‐18, as seen in Apc^Min/+ × fat‐1 mice. We conclude that ω‐3PUFAs can prevent intestinal polyp formation by inhibition of Wnt/β‐catenin signaling, but increased levels of 15‐PGDH and IL‐18.