Contribution of xanthine oxidoreductase to mammary epithelial and breast cancer cell differentiation in part modulates inhibitor of differentiation-1

Loss of xanthine oxidoreductase (XOR) has been linked to aggressive breast cancer in vivo and to breast cancer cell aggressiveness in vitro. In the present study, we hypothesized that the contribution of XOR to the development of the normal mammary gland may underlie its capacity to modulate breast cancer. We contrasted in vitro and in vivo developmental systems by differentiation marker and microarray analyses. Human breast cancer microarray was used for clinical outcome studies. The role of XOR in differentiation and proliferation was examined in human breast cancer cells and in a mouse xenograft model. Our data show that XOR was required for functional differentiation of mammary epithelial cells both in vitro and in vivo. Poor XOR expression was observed in a mouse ErbB2 breast cancer model, and pharmacologic inhibition of XOR increased breast cancer tumor burden in mouse xenograft. mRNA microarray analysis of human breast cancer revealed that low XOR expression was significantly associated with time to tumor relapse. The opposing expression of XOR and inhibitor of differentiation-1 (Id1) during HC11 differentiation and mammary gland development suggested a potential functional relationship. While overexpression of Id1 inhibited HC11 differentiation and XOR expression, XOR itself modulated expression of Id1 in differentiating HC11 cells. Overexpression of XOR both inhibited Id1-induced proliferation and -stimulated differentiation of Heregulin-β1-treated human breast cancer cells. These results show that XOR is an important functional component of differentiation whose diminished expression contributes to breast cancer aggressiveness, and they support XOR as both a breast cancer biomarker and a target for pharmacologic activation in therapeutic management of aggressive breast cancer.     

Claudin-16 reduces the aggressive behavior of human breast cancer cells

Claudin-16 (Paracellin-1) is a transmembrane tight junction (TJ) protein originally described as having a critical role in the re-absorption of magnesium and calcium in the kidney. This study examined expression of Claudin-16 in human breast cells and tissues to identify a possible link between expression and aggressiveness in cells and between Claudin-16 levels and patient prognosis. Insertion of the Claudin-16 gene into MDA-MB-231 human breast cancer cells resulted in cells that were significantly less motile and invasive in behavior, with increased adhesion to matrix. These cells also exhibited significantly increased TJ functionality and "tighter" colony morphology. Moreover, growth rates were reduced in both in vitro and in vivo assays (P < 0.002). Frozen sections from breast cancer primary tumors (matched tumor 124 and background 33) were immuno-stained. RNA was reverse transcribed and analyzed by Q-PCR (standardized using beta-actin, normalized with cytokeratin-19 levels). Levels of expression of Claudin-16 were significantly decreased in node positive tumors compared to negative (P = 0.016). Expression was significantly lower in patients with node positive tumors (P = 0.016) and in those who had died from breast cancer or had general poor prognosis (P < 0.015). Immunohistochemical staining showed decreased expression of Claudin-16 in tumor sections (P < 0.00001). In conclusion, forced expression of Claudin-16 in breast cancer cells resulted in a less aggressive phenotype and reduced in vivo tumor volume. Claudin-16 expression was reduced in human breast cancer, particularly in patients with aggressive tumors and high mortality. This suggests that Claudin-16 plays a role beyond that of an initial metastasis repressor in this cancer type.     

Functional analysis of Csk and CHK kinases in breast cancer cells.

In this report, we analyzed the expression and kinase activities of Csk and CHK kinases in normal breast tissues and breast tumors and their involvement in HRG-mediated signaling in breast cancer cells. Csk expression and kinase activity were abundant in normal human breast tissues, breast carcinomas, and breast cancer cell lines, whereas CHK expression was negative in normal breast tissues and low in some breast tumors and in the MCF-7 breast cancer cell line. CHK kinase activity was not detected in human breast carcinoma tissues (12 of 12) or in the MCF-7 breast cancer cell line (due to the low level of CHK protein expression), but was significantly induced upon heregulin (HRG) stimulation. We have previously shown that CHK associates with the ErbB-2/neu receptor upon HRG stimulation via its SH2 domain and that it down-regulates the ErbB-2/neu-activated Src kinases. Our new findings demonstrate that Csk has no effect on ErbB-2/neu-activated Src kinases upon HRG treatment and that its kinase activity is not modulated by HRG. CHK significantly inhibited in vitro cell growth, transformation, and invasion induced upon HRG stimulation. In addition, tumor growth of wt CHK-transfected MCF-7 cells was significantly inhibited in nude mice. Furthermore, CHK down-regulated c-Src and Lyn protein expression and kinase activity, and the entry into mitosis was delayed in the wt CHK-transfected MCF-7 cells upon HRG treatment. These results indicate that CHK, but not Csk, is involved in HRG-mediated signaling pathways, down-regulates ErbB-2/neu-activated Src kinases, and inhibits invasion and transformation of breast cancer cells upon HRG stimulation. These findings strongly suggest that CHK is a novel negative growth regulator of HRG-mediated ErbB-2/neu and Src family kinase signaling pathways in breast cancer cells.     

Notch1 Is a 5-Fluorouracil Resistant and Poor Survival Marker in Human Esophagus Squamous Cell Carcinomas

Notch signaling involves the processes that govern cell proliferation, cell fate decision, cell differentiation and stem cell maintenance. Due to its fundamental role in stem cells, it has been speculated during the recent years that Notch family may have critical functions in cancer stem cells or cancer cells with a stem cell phenotype, therefore playing an important role in the process of oncogenesis. In this study, expression of Notch family in KYSE70, KYSE140 and KYSE450 squamous esophageal cancer cell lines and virus transformed squamous esophageal epithelial cell line Het-1A was examined by quantitative RT-PCR. Compared to the Het-1A cells, higher levels of Nocth1 and Notch3 expression in the cancer cell lines were identified. Due to the finding that NOTCH3 mainly mediates squamous cell differentiation, NOTCH1 expression was further studied in these cell lines. By Western blot analyses, the KYSE70 cell line which derived from a poorly differentiated tumor highly expressed Notch1, and the Notch1 expression in this cell line was hypoxia inducible, while the KYSE450 cell line which derived from a well differentiated tumor was always negative for Notch1, even in hypoxia. Additional studies demonstrated that the KYSE70 cell line was more 5-FU resistant than the KYSE450 cell line and such 5-FU resistance is correlated to Notch1 expression verified by Notch1 knockdown experiments. In clinical samples, Notch1 protein expression was detected in the basal cells of human esophagus epithelia, and its expression in squamous cell carcinomas was significantly associated with higher pathological grade and shorter overall survival. We conclude that Notch1 expression is associated with cell aggressiveness and 5-FU drug resistance in human esophageal squamous cell carcinoma cell lines in vitro and is significantly associated with a poor survival in human esophageal squamous cell carcinomas.     

Upregulation of HYAL1 expression in breast cancer promoted tumor cell proliferation, migration, invasion and angiogenesis

Hyaluronic acid (HA) is a component of the Extra-cellular matrix (ECM), it is closely correlated with tumor cell growth, proliferation, metastasis and angiogenesis, etc. Hyaluronidase (HAase) is a HA-degrading endoglycosidase, levels of HAase are elevated in many cancers. Hyaluronidase-1 (HYAL1) is the major tumor-derived HAase. We previously demonstrated that HYAL1 were overexpression in human breast cancer. Breast cancer cells with higher HAase expression, exhibited significantly higher invasion ability through matrigel than those cells with lower HAase expression, and knockdown of HYAL1 expression in breast cancer cells resulted in decreased cell growth, adhesion, invasion and angiogenesis. Here, to further elucidate the function of HYAL1 in breast cancer, we investigated the consequences of forcing HYAL1 expression in breast cancer cells by transfection of expression plasmid. Compared with control, HYAL1 up-regulated cells showed increased the HAase activity, and reduced the expression of HA in vitro. Meantime, upregulation of HYAL1 promoted the cell growth, migration, invasion and angiogenesis in vitro. Moreover, in nude mice model, forcing HYAL1 expression induced breast cancer cell xenograft tumor growth and angiogenesis. Interestingly, the HA expression was upregulated by forcing HYAL1 expression in vivo. These findings suggested that HYAL1-HA system is correlated with the malignant behavior of breast cancer.     

Profiling and comparative analysis of glycoproteins in Hs578BST and Hs578T and investigation of prolyl 4-Hydroxylase alpha polypeptide II expression and influence in breast cancer cells

To identify potential cancer related glycoproteins in breast cancer cells, we enriched N-linked glycoproteins by lentil lectin from the human breast cancer cell line Hs578T and the normal breast cell line Hs578BST for proteomic comparison. Glycoproteins were separated and compared by two-dimensional electrophoresis. Twenty-four glycoproteins were identified that expressed remarkably differently, among which nine were involved in the progress of collagen synthesis. Prolyl 4-hydroxylase alpha polypeptide II (P4HA2) expression and influence in breast cancer was further investigated. Immunohistochemistry revealed that P4HA2 was upregulated in breast tumor cells compared with its adjacent normal tissues. Moreover, overexpression and RNA interference of P4HA2 showed that P4HA2 expression suppressed cell proliferation and migration in Hs578T in vitro.     

Expression of transforming growth factor alpha and its messenger ribonucleic acid in human breast cancer: its regulation by estrogen and its possible functional significance

We have studied the estrogenic regulation and the potential autocrine role of transforming growth factor alpha (TGF alpha) in the human breast cancer cell line MCF-7. A biologically active apparent mol wt 30 k TGF alpha was identified by gel filtration chromatography in medium conditioned by MCF-7 breast cancer cells. We previously reported induction of TGF alpha levels in medium by 17 beta-estradiol. We now report correlated increases in TGF alpha mRNA, by Northern and slot blot analysis, after estrogen treatment of MCF-7 cells in vitro. In vivo experiments confirmed these data: estrogen withdrawal from MCF-7 tumor-bearing nude mice resulted in a decline in tumor size and TGF alpha mRNA levels. To explore the functional significance of TGF alpha in MCF-7 cells, anti-TGF alpha antibody was added to MCF-7 soft agar cloning assays. Inhibition of MCF-7 growth resulted, supporting an autocrine role for TGF alpha. Further experiments using an anti-EGF receptor antibody expanded this data, demonstrating inhibition of estrogen-stimulated monolayer MCF-7 cell growth. Examining the generality of TGF alpha expression, 4.8 kilobase TGF alpha mRNAs were seen in three other human breast cancer cell lines, MDA-MB-231, ZR 75B, and T47D. Expression of TGF alpha mRNA was detected in 70% of estrogen receptor positive and negative primary human breast tumors from 40 patients when examined by slot blot and Northern analysis. Thus, we have demonstrated broad expression of TGF alpha in human breast cancer, its hormonal regulation in an estrogen-responsive cell line, and its possible functional significance in MCF-7 cell growth.     

Angiopoietin-2, an angiogenic regulator, promotes initial growth and survival of breast cancer metastases to the lung through the integrin-linked kinase (ILK)-AKT-B cell lymphoma 2 (Bcl-2) pathway

The early onsets of breast cancer metastasis involve cell retention, survival, and resistant to apoptosis and subsequent growth at target vascular beds and tissues in distant organs. We previously reported that angiopoietin-2 (Ang2), an angiogenic regulator stimulates MCF-7 breast tumor metastasis from their orthotopic sites to distant organs through the α(5)β(1) integrin/integrin-linked kinase (ILK)/Akt pathway. Here, by using an experimental tumor metastasis model and in vitro studies, we further dissect the underlying mechanism by which Ang2 promotes the initial growth and survival of MCF-7 breast cancer metastasis in the lung of animals. We show that Ang2 increases cell survival and suppresses cell apoptosis through ILK-induced phosphorylation of Akt1, Akt2, and up-regulation of Bcl-2 in breast cancer cells. Inhibition of ILK, Akt1, and Akt2, and their effector Bcl-2 diminishes Ang2-stimulated breast cancer cell survival and Ang2-attenuated apoptosis in vitro, and initial survival and growth of breast cancer metastasis in the lung of animals. Additionally, siRNA knockdown of endogenous Ang2 in three human metastatic breast cancer cell lines also inhibits phosphorylation of Akt, expression of Bcl-2, and tumor cell survival, migration, and increases cell apoptosis. Since increased expression of Ang2 correlates with elevated potential of human breast cancer metastasis in clinic, our data underscore the importance that up-regulated Ang2 not only stimulates breast cancer growth and metastasis at late stages of the process, but is also critical at the initiating stages of metastases onset, thereby suggesting Ang2 as a promising therapeutic target for treating patients with metastatic breast cancer.     

H-Ras-specific upregulation of granulocyte colony-stimulating factor promotes human breast cell invasion via matrix metalloproteinase-2

Ras expression has been suggested to be a marker for tumor aggressiveness of breast cancer. We previously showed that H-Ras, but not N-Ras, induced invasive/migratory phenotypes in MCF10A human breast epithelial cells. The present study aimed to determine the role of granulocyte colony-stimulating factor in H-Ras-induced malignant progression of human breast epithelial cells. Here, we show that G-CSF plays a crucial role in H-Ras-induced MCF10A cell invasion and migration. The siRNA-mediated knockdown of G-CSF significantly reduced H-Ras-induced matrix metalloproteinase (MMP)-2 expression, as well as invasion/migration, suggesting the functional significance of G-CSF in the invasive phenotype of human breast cells. Importantly, the induction of G-CSF expression conferred the invasive/migratory phenotypes to MCF10A cells with up-regulation of MMP-2 and activation of Rac1, MKK3/6, p38 MAPK, Akt, and ERKs. Knockdown of Rac1 by siRNA significantly inhibited MMP-2 upregulation and invasiveness of G-CSF MCF10A cells, demonstrating that G-CSF-induced MMP-2 upregulation and invasive phenotype is mediated by Rac1. Using human breast tissues and sera from breast cancer patients, we further demonstrate that the expression level of G-CSF is strongly correlated with pathologically-diagnosed breast cancer. These data provide a molecular basis for the crucial role of G-CSF in promoting invasiveness of human breast epithelial cells.     

Nicotinamide phosphoribosyltransferase can affect metastatic activity and cell adhesive functions by regulating integrins in breast cancer

NAD⁺ metabolism is an essential regulator of cellular redox reactions, energy pathways, and a substrate provider for NAD⁺ consuming enzymes. We recently demonstrated that enhancement of NAD⁺/NADH levels in breast cancer cells with impaired mitochondrial NADH dehydrogenase activity, through augmentation of complex I or by supplementing tumor cell nutrients with NAD⁺ precursors, inhibits tumorigenicity and metastasis. To more fully understand how aberrantly low NAD⁺ levels promote tumor cell dissemination, we here asked whether inhibition of NAD⁺ salvage pathway activity by reduction in nicotinamide phosphoribosyltransferase (NAMPT) expression can impact metastasis and tumor cell adhesive functions. We show that knockdown of NAMPT, the enzyme catalyzing the rate-limiting step of the NAD⁺ salvage pathway, enhances metastatic aggressiveness in human breast cancer cells and involves modulation of integrin expression and function. Reduction in NAMPT expression is associated with upregulation of select adhesion receptors, particularly αvβ3 and β1 integrins, and results in increased breast cancer cell attachment to extracellular matrix proteins, a key function in tumor cell dissemination. Interestingly, NAMPT downregulation prompts expression of integrin αvβ3 in a high affinity conformation, known to promote tumor cell adhesive interactions during hematogenous metastasis. NAMPT has been selected as a therapeutic target for cancer therapy based on the essential functions of this enzyme in NAD⁺ metabolism, cellular redox, DNA repair and energy pathways. Notably, our results indicate that incomplete inhibition of NAMPT, which impedes NAD⁺ metabolism but does not kill a tumor cell can alter its phenotype to be more aggressive and metastatic. This phenomenon could promote cancer recurrence, even if NAMPT inhibition initially reduces tumor growth.     

Beta(3) integrin expression increases breast carcinoma cell responsiveness to the malignancy-enhancing effects of osteopontin

Osteopontin (OPN) is a secreted phosphoprotein that has been associated with malignancy of breast and other cancers. OPN binds to several cell surface integrins including alpha(v)beta(3), alpha(v)beta(5), and alpha(v)beta(1). Although the relative contribution of these integrins to breast cancer cell malignancy is uncertain, correlative studies suggest that alpha(v)beta(3) may be particularly associated with increased tumor aggressiveness. Previously, we reported that tumorigenic, nonmetastatic 21NT mammary carcinoma cells respond to OPN through alpha(v)beta(5) and alpha(v)beta(1) but not alpha(v)beta(3). Here, we determined that 21NT cells lack beta(3) expression, and we asked whether expression of alpha(v)beta(3) could enhance the ability of breast cancer cells to respond to the malignancy-promoting effects of OPN both in vitro and in vivo. 21NT cells stably transfected with beta(3) showed significantly increased adhesion, migration, and invasion to OPN in vitro compared with vector control. To determine if beta(3) could also enhance the response of breast epithelial cells to OPN in vivo, cells stably transfected with both beta(3) and OPN (NT/Obeta(3)) were injected into the mammary fat pad of female nude mice and primary tumor growth was assessed relative to controls. Mice injected with NT/Obeta(3) cells demonstrated a significantly increased primary tumor take (75% of mice) compared with controls (0-12.5% of mice) as well as a decreased tumor doubling time and a decreased tumor latency period. These results suggest that increased expression of the alpha(v)beta(3) integrin during breast cancer progression can make tumor cells more responsive to malignancy-promoting ligands such as OPN and result in increased tumor cell aggressiveness.     

Role of KCNMA1 in Breast Cancer

KCNMA1 encodes the α-subunit of the large conductance, voltage and Ca²⁺-activated (BK) potassium channel and has been reported as a target gene of genomic amplification at 10q22 in prostate cancer. To investigate the prevalence of the amplification in other human cancers, the copy number of KCNMA1 was analyzed by fluorescence-in-situ-hybridization (FISH) in 2,445 tumors across 118 different tumor types. Amplification of KCNMA1 was restricted to a small but distinct fraction of breast, ovarian and endometrial cancer with the highest prevalence in invasive ductal breast cancers and serous carcinoma of ovary and endometrium (3–7%). We performed an extensive analysis on breast cancer tissue microarrays (TMA) of 1,200 tumors linked to prognosis. KCNMA1 amplification was significantly associated with high tumor stage, high grade, high tumor cell proliferation, and poor prognosis. Immunofluorescence revealed moderate or strong KCNMA1 protein expression in 8 out of 9 human breast cancers and in the breast cancer cell line MFM223. KCNMA1-function in breast cancer cell lines was confirmed by whole-cell patch clamp recordings and proliferation assays, using siRNA-knockdown, BK channel activators such as 17ß-estradiol and the BK-channel blocker paxilline. Our findings revealed that enhanced expression of KCNMA1 correlates with and contributes to high proliferation rate and malignancy of breast cancer.     

Osteoactivin promotes breast cancer metastasis to bone

The skeleton is a preferred site of metastasis in patients with disseminated breast cancer. We have used 4T1 mouse mammary carcinoma cells, which metastasize to bone from the mammary fat pads of immunocompetent mice, to identify novel genes involved in this process. In vivo selection of parental cells resulted in the isolation of independent, aggressively bone metastatic breast cancer populations with reduced metastasis to the lung. Gene expression profiling identified osteoactivin as a candidate that is highly and selectively expressed in aggressively bone metastatic breast cancer cells. These cells displayed enhanced migratory and invasive characteristics in vitro, the latter requiring sustained osteoactivin expression. Osteoactivin depletion in these cells, by small interfering RNA, also lead to a loss of matrix metalloproteinase-3 expression, whereas forced osteoactivin expression in parental 4T1 cells was sufficient to elevate matrix metalloproteinase-3 levels, suggesting that this matrix metalloproteinase may be an important mediator of osteoactivin function. Overexpression of osteoactivin in an independent, weakly bone metastatic breast cancer cell model significantly enhanced the formation of osteolytic bone metastases in vivo. Finally, high levels of osteoactivin expression in primary human breast cancers correlate with estrogen receptor-negative status and increasing tumor grade. Thus, we have identified osteoactivin as a protein that is expressed in aggressive human breast cancers and is capable of promoting breast cancer metastasis to bone.     

Lin28 mediates paclitaxel resistance by modulating p21, Rb and Let-7a miRNA in breast cancer cells

Resistance to chemotherapy is a major obstacle for the effective treatment of cancers. Lin28 has been shown to contribute to tumor relapse after chemotherapy; however, the relationship between Lin28 and chemoresistance remained unknown. In this study, we investigated the association of Lin28 with paclitaxel resistance and identified the underlying mechanisms of action of Lin28 in human breast cancer cell lines and tumor tissues. We found that the expression level of Lin28 was closely associated with the resistance to paclitaxel treatment. The T47D cancer cell line, which highly expresses Lin28, is more resistant to paclitaxel than the MCF7, Bcap-37 or SK-BR-3 cancer cell lines, which had low-level expression of Lin28. Knocking down of Lin28 in Lin28 high expression T47D cells increased the sensitivity to paclitaxel treatment, while stable expression of Lin28 in breast cancer cells effectively attenuated the sensitivity to paclitaxel treatment, resulting in a significant increase of IC50 values of paclitaxel. Transfection with Lin28 also significantly inhibited paclitaxel-induced apoptosis. We also found that Lin28 expression was dramatically increased in tumor tissues after neoadjuvant chemotherapy or in local relapse or metastatic breast cancer tissues. Moreover, further studies showed that p21, Rb and Let-7 miRNA were the molecular targets of Lin28. Overexpression of Lin28 in breast cancer cells considerably induced p21 and Rb expression and inhibited Let-7 miRNA levels. Our results indicate that Lin28 expression might be one mechanism underlying paclitaxel resistance in breast cancer, and Lin28 could be a potential target for overcoming paclitaxel resistance in breast cancer.     

Expression of NgBR Is Highly Associated with Estrogen Receptor Alpha and Survivin in Breast Cancer

NgBR is a type I receptor with a single transmembrane domain and was identified as a specific receptor for Nogo-B. Our recent findings demonstrated that NgBR binds farnesylated Ras and recruits Ras to the plasma membrane, which is a critical step required for the activation of Ras signaling in human breast cancer cells and tumorigenesis. Here, we first use immunohistochemistry and real-time PCR approaches to examine the expression patterns of Nogo-B and NgBR in both normal and breast tumor tissues. Then, we examine the relationship between NgBR expression and molecular subtypes of breast cancer, and the roles of NgBR in estrogen-dependent survivin signaling pathway. Results showed that NgBR and Nogo-B protein were detected in both normal and breast tumor tissues. However, the expression of Nogo-B and NgBR in breast tumor tissue was much stronger than in normal breast tissue. The statistical analysis demonstrated that NgBR is highly associated with ER-positive/HER2-negative breast cancer. We also found that the expression of NgBR has a strong correlation with the expression of survivin, which is a well-known apoptosis inhibitor. The correlation between NgBR and survivin gene expression was further confirmed by real-time PCR. In vitro results also demonstrated that estradiol induces the expression of survivin in ER-positive T47D breast tumor cells but not in ER-negative MDA-MB-468 breast tumor cells. NgBR knockdown with siRNA abolishes estradiol-induced survivin expression in ER-positive T47D cells but not in ER-negative MDA-MB-468 cells. In addition, estradiol increases the expression of survivin and cell growth in ER-positive MCF-7 and T47D cells whereas knockdown of NgBR with siRNA reduces estradiol-induced survivin expression and cell growth. In summary, these results indicate that NgBR is a new molecular marker for breast cancer. The data suggest that the expression of NgBR may be essential in promoting ER-positive tumor cell proliferation via survivin induction in breast cancer.     

Specific expression of the human voltage-gated proton channel Hv1 in highly metastatic breast cancer cells, promotes tumor progression and metastasis

The newly discovered human voltage-gated proton channel Hv1 is essential for proton transfer, which contains a voltage sensor domain (VSD) without a pore domain. We report here for the first time that Hv1 is specifically expressed in the highly metastatic human breast tumor tissues, but not in poorly metastatic breast cancer tissues, detected by immunohistochemistry. Meanwhile, real-time RT-PCR and immunocytochemistry showed that the expression levels of Hv1 have significant differences among breast cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3, in which Hv1 is expressed at a high level in highly metastatic human breast cancer cell line MDA-MB-231, but at a very low level in poorly metastatic human breast cancer cell line MCF-7. Inhibition of Hv1 expression in the highly metastatic MDA-MB-231 cells by small interfering RNA (siRNA) significantly decreases the invasion and migration of the cells. The intracellular pH of MDA-MB-231 cells down-regulated Hv1 expression by siRNA is obviously decreased compared with MDA-MB-231 with the scrambled siRNA. The expression of matrix metalloproteinase-2 and gelatinase activity in MDA-MB-231 cells suppressed Hv1 by siRNA were reduced. Our results strongly suggest that Hv1 regulates breast cancer intracellular pH and exacerbates the migratory ability of metastatic cells.     

In situ expression of IFN-γ-inducible T cell α chemoattractant in breast cancer mounts an enhanced specific anti-tumor immunity which leads to tumor regression

Increased evidence indicates that chemokines are involved in tumor growth. ITAC, a key member of chemokines, possesses the ability to recruit T cells and enhance immune responses. Therefore, ITAC might contribute to antitumor immunity. In this study, we evaluated the relationship between the expression of ITAC and human breast cancer advancement. We further investigated whether forced expression of ITAC in tumor sites could mediate enhanced antitumor immunity in a murine breast cancer model. Results showed that ITAC expression level was down-regulated in 31 breast cancer specimens compared to normal mammary tissues, and associated negatively with the stages of breast cancer. Contrarily, forced expression of ITAC in murine 4T1 tumor cells resulted in tumor regression after initial growth upon injection into naïve Balb/c mice. More lymphocytes were recruited to the site of tumor inoculated by 4T1-ITAC and more than 80% of these T cells expressed the ITAC receptor, CXCR3. ITAC-recruited TILs exhibited 4T1-specific proliferation and cytotoxicity, and an increased IFN-γ but decreased IL-4 production. Importantly, forced expression of ITAC in 4T1 tumor nodules inhibited tumor growth. These findings demonstrated that the decreased expression of ITAC is associated with the advancement of breast cancer in patients. Forced expression of ITAC in tumor site not only induces increased T cell-recruitment and elicits a specific antitumor immunity, but also mediates regression of established 4T1 tumors, indicating the potential application of ITAC-expressing tumor cells in cancer immunotherapy and vaccine designing.