Localization In Breast Cancer Cells Research Articles

Abstract PO4-24-03: Adiponectin modulates cell polarity according to ERα expression in breast cancer cells

Abstract INTRODUCTION: Tumor microenvironment (TME) is a complex and heterogeneous network, consisting of stromal and immune cells, embedded with cytokines, growth factors, soluble receptors, and exosomes. Mutual and dynamic crosstalk among cancer, stromal and immune cells predispose cancer cells to metastasis. Particularly, the main component of breast TME is represented by adipocytes, which secretory activity is compromised in obesity. Indeed, hypertrophic, and hyperplastic adipocytes undergo to a modified cytokines secretory pattern, characterized by a reduced secretion of Adiponectin, now recognized as a crucial factor in the pathogenesis of breast cancer. LKB1 is one the most important effector of Adiponectin transduction pathways, and recently it has been demonstrated its role as ERα coactivator in promoting breast cancer cell growth. Moreover, LKB1 is a key regulator in controlling cell polarity along with E-cadherin and Cdc42. Cell polarity has been considered essential for mammary epithelial integrity, and disruption of individual polarity proteins is sufficient to induce tumor progression. The aim of the present study was to investigate the effects of adiponectin on the regulation and alteration of cell polarity, essential for breast cancer cell growth and invasiveness. METHODS: Immunofluorescence analyses were done to detect protein localization in breast cancer cells. Protein expression was performed by immunoblotting in the presence or absence of ERa and LKB1 siRNA. LKB1/Cdc42 and LKB1/E-cadherin interaction was detected by Proximity ligation and immunoprecipitation assay respectively. Wound healing assay was carried out to evaluate GM130 orientation. Growth and metastatic potential of breast cancer cells were assessed in vivo by orthotopic model and metastatic study respectively. RESULTS: In Adiponectin-treated MCF-7 cells LKB1 is mostly present in the nucleus, recruited as ERa-coactivator, while in BT20 cells LKB1 is localized with E-cadherin in the cytosolic compartment, suggesting its role in polarity. Our results showed that in MCF-7 cells Adiponectin increases Cdc42 level depending on LKB1 expression. Moreover, Cdc42 and LKB1 interact and colocalized in the nucleus of Adiponectin-treated MCF-7 cells. On the contrary, LKB1 and Cdc42 are located with E-cadherin in the cytosol of BT20 cells, addressing their cytosolic cooperation in maintaining cell polarity, as confirmed by an increased Cdc42 activity. Immunofluorescence analyses demonstrated a lack of Golgi realignment toward the front of the leading edge in MCF-7 cells treated with Adiponectin, not observed in BT20 cells. All this suggest a loss of cell polarity organization that predispose to cancer growth and progression, as confirmed by an increased tumor volume mass and a major number of lung metastatic events upon tail vein injection of adiponectin-treated ERα-positive cells with respect to untreated cells. CONCLUSION: In ERα-positive breast cancer cells Adiponectin modulates the expression of the main proteins involved in cell polarity, leading to increased growth, progression, and distant metastasis. Citation Format: Giuseppina Daniela Naimo, Martina Forestiero, Alessandro Paolì, Luca Gelsomino, Rocco Malivindi, Adele Elisabetta Leonetti, Francesca Giordano, Maria Luisa Panno, Loredana Mauro, Sebastiano Andò. Adiponectin modulates cell polarity according to ERα expression in breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-03.

Abstract 3633: Exploring a novel crosstalk between hyluronan/CD44 and ErbB-2 pathways in resistance to ErbB-2-targeted therapies in breast cancer

Abstract The tumor microenvironment component hyaluronan (HA) induces tumor progression through the interaction with its cell surface receptor CD44. Accumulation of HA is associated with poor prognosis and resistance to the anti-ErbB-2 agent trastuzumab (TZ) in breast cancer (BC). ErbB-2 receptor is overexpressed in 15-20% of BC patients (ErbB-2+) and constitutes an important therapeutic target. Despite the clinical efficiency of therapies targeting ErbB-2, resistance to these drugs remains a major issue. In addition to its membrane function, ErbB-2 migrates to the nucleus (NErbB-2) where it acts as a transcription factor (TF) or as a coactivator of TF, modulating proliferation, metastasis, and resistance to anti-ErbB-2 therapies in BC. CD44 has also been found in the nucleus (NCD44). Although crosstalk between HA/CD44 and ErbB-2 pathways has been reported, how their molecular interactions mediate TZ resistance remains poorly known. Our in silico studies showed that TZ-resistant cells express higher CD44 levels than TZ-sensitive ones. We previously reported that stimulation with the ErbBs ligand heregulin (HRG) induces NErbB-2 translocation, acquired-TZ resistance and proliferation in the TZ-sensitive SK-BR-3 cell line. Here, we found that HRG also increased CD44 mRNA expression in SK-BR-3 cells. Then, we explored the role of HA in modulating CD44 and ErbB-2 nuclear localization in BC cells. Immunofluorescence and confocal microscopy studies showed that stimulation with exogenous HA induced nuclear translocation of ErbB-2 in T47D cells. Interestingly, we found constitutive presence of CD44 and ErbB-2 protein in nuclear lysates of JIMT-1 cells, a de novo TZ-resistant BC model. HA stimuli further enhanced this nuclear localization. Contrarily, treatment with the chemical inhibitor of HA synthesis 4-methylumbelliferone (4MU) decreased not only HA levels but also NErbB-2 in JIMT-1 cells. Furthermore, 4MU inhibited the proliferation of two TZ-resistant cell lines, JIMT-1 and MDA-MB-453, in a dose-dependent manner. 4MU also inhibited HRG-induced proliferation in SK-BR-3 cells. Even more, wound-healing assays showed that 4MU inhibited migration of JIMT-1 cells. This inhibition was similar to the one observed when ErbB-2 was excluded from the nucleus via transfection with the hErbB-2ΔNLS mutant. In summary, we reveal that HA induces CD44 and ErbB-2 nuclear localization in BC cells, suggesting a novel crosstalk between HA/CD44 and ErbB-2 pathways. These findings also highlight the blockade of HA synthesis with 4MU as a novel therapeutic strategy in TZ-resistant BC. Citation Format: Santiago Madera, Valentina C. Bulian, Sharon S. Merin, María F. Mercogliano, Roxana Schillaci, Rosalia I. Cordo Russo. Exploring a novel crosstalk between hyluronan/CD44 and ErbB-2 pathways in resistance to ErbB-2-targeted therapies in breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3633.

CD44 Promotes Breast Cancer Metastasis through AKT-Mediated Downregulation of Nuclear FOXA2.

The primary cause of breast cancer mortality is the metastatic invasion of cancerous stem cells (CSC). Cluster of differentiation 44 (CD44) is a well-known CSC marker in various cancers, as well as a key role player in metastasis and relapse of breast cancer. CD44 is a cell-membrane embedded protein, and it interacts with different proteins to regulate cancer cell behavior. Transcription factor forkhead box protein A2 (FOXA2) acts as an important regulator in multiple cancers, including breast cancer. However, the biological significance of CD44-FOXA2 association in breast cancer metastasis remains unclear. Herein, we observed that CD44 expression was higher in metastatic lymph nodes compared to primary tumors using a flow cytometric analysis. CD44 overexpression in breast cancer cell lines significantly promoted cell migration and invasion abilities, whereas the opposite effects occurred upon the knockdown of CD44. The stem cell array analysis revealed that FOXA2 expression was upregulated in CD44 knockdown cells. However, the knockdown of FOXA2 in CD44 knockdown cells reversed the effects on cell migration and invasion. Furthermore, we found that CD44 mediated FOXA2 localization in breast cancer cells through the AKT pathway. Moreover, the immunofluorescence assay demonstrated that AKT inhibitor wortmannin and AKT activator SC79 treatment in breast cancer cells impacted FOXA2 localization. Collectively, this study highlights that CD44 promotes breast cancer metastasis by downregulating nuclear FOXA2.

ICAM-1 Targeted Drug Combination Nanoparticles Enhanced Gemcitabine-Paclitaxel Exposure and Breast Cancer Suppression in Mouse Models.

Despite the availability of molecularly targeted treatments such as antibodies and small molecules for human epidermal growth factor receptor 2 (HER2), hormone receptor (HR), and programmed death-ligand 1 (PD-L1), limited treatment options are available for advanced metastatic breast cancer (MBC), which constitutes ~90% mortality. Many of these monotherapies often lead to drug resistance. Novel MBC-targeted drug-combination therapeutic approaches that may reduce resistance are urgently needed. We investigated intercellular adhesion molecule-1 (ICAM-1), which is abundant in MBC, as a potential target to co-localize two current drug combinations, gemcitabine (G) and paclitaxel (T), assembled in a novel drug-combination nanoparticle (GT DcNP) form. With an ICAM-1-binding peptide (referred to as LFA1-P) coated on GT DcNPs, we evaluated the role of the LFA1-P density in breast cancer cell localization in vitro and in vivo. We found that 1–2% LFA1-P peptide incorporated on GT DcNPs provided optimal cancer cell binding in vitro with ~4× enhancement compared to non-peptide GT DcNPs. The in vivo probing of GT DcNPs labeled with a near-infrared marker, indocyanine green, in mice by bio-imaging and G and T analyses indicated LFA1-P enhanced drug and GT DcNP localization in breast cancer cells. The target/healthy tissue (lung/gastrointestinal (GI)) ratio of particles increased by ~60× compared to the non-ligand control. Collectively, these data indicated that LFA1 on GT DcNPs may provide ICAM-1-targeted G and T drug combination delivery to advancing MBC cells found in lung tissues. As ICAM-1 is generally expressed even in breast cancers that are triple-negative phenotypes, which are unresponsive to inhibitors of nuclear receptors or HER2/estrogen receptor (ER) agents, ICAM-1-targeted LFA1-P-coated GT DcNPs should be considered for clinical development to improve therapeutic outcomes of MBCs.

IRSp53 is a novel interactor of SHIP2: A role of the actin binding protein Mena in their cellular localization in breast cancer cells

A tight control of the machineries regulating membrane bending and actin dynamics is very important for the generation of membrane protrusions, which are crucial for cell migration and invasion. Protein/protein and protein/phosphoinositides complexes assemble and disassemble to coordinate these mechanisms, the scaffold properties of the involved proteins playing a prominent role in this organization. The PI 5-phosphatase SHIP2 is a critical enzyme modulating PI(3,4,5)P3, PI(4,5)P2 and PI(3,4)P2 content in the cell. The scaffold properties of SHIP2 contribute to the specific targeting or retention of the protein in particular subcellular domains. Here, we identified IRSp53 as a new binding interactor of SHIP2 proline-rich domain. Both proteins are costained in HEK293T cells protrusions, upon transfection. We showed that the SH3-binding polyproline motif recognized by IRSp53 in SHIP2 is different from the regions targeted by other PRR binding partners i.e., CIN85, ITSN or even Mena a common interactor of both SHIP2 and IRSp53. We presented evidence that IRSp53 phosphorylation on S366 did not influence its interaction with SHIP2 and that Mena is not necessary for the association of SHIP2 with IRSp53 in MDA-MB-231 cells. The absence of Mena in MDA-MB-231 cells decreased the intracellular content in F-actin and modified the subcellular localization of SHIP2 and IRSp53 by increasing their relative content at the plasma membrane. Together our data suggest that SHIP2, through interaction with the cell protrusion regulators IRSp53 and Mena, participate to the formation of multi-protein complexes. This ensures the appropriate modulations of PIs which is important for regulation of membrane dynamics.

Critical Role of Estrogen Receptor Alpha O-Glycosylation by N-Acetylgalactosaminyltransferase 6 (GALNT6) in Its Nuclear Localization in Breast Cancer Cells

Alteration of protein O-glycosylation in various human cancers including breast cancer is well known, but molecular roles of their aberrant glycosylations on cancer have not been fully understood. We previously reported critical roles of polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6 or GalNAc-T6) that was upregulated in a great majority of breast cancer tissues. Here we further report O-glycosylation of estrogen receptor alpha (ER-α) by GALNT6 and the significant role of its nuclear localization in breast cancer cells. Knockdown of GALNT6 expression in two breast cancer cell lines, T47D and MCF7, in which both ER-α and GALNT6 were highly expressed, by small interfering RNA could significantly attenuate expression of ER-α. Immunocytochemical analysis clearly demonstrated the drastic decrease of ER-α protein in the nucleus of these cancer cells. Accordingly, the downstream genes of the ER-α pathway such as MYC, CCND1, and CTSD were significantly downregulated. We confirmed GALNT6-dependent ER-α O-glycosylation and identified O-glycosylation of S573 in an F domain of ER-α by GALNT6 through LC-MS/MS analysis. We also obtained evidences showing that the glycosylation of ER-α at S573 by GALNT6 is essential for protein stability and nuclear localization of ER-α in breast cancer cells. Furthermore, we designed cell membrane–permeable peptides including the O-glycosylation site and found a significant decrease of the cell viability of breast cancer cells by treatment of these peptides in a GALNT6 expression–dependent manner. Our study suggests that targeting the GALNT6 enzymatic activity as well as the GALNT6/ER-α interaction could be a promising therapeutic approach to ER-α–positive breast cancer patients.

Dysregulation of POPDC1 promotes breast cancer cell migration and proliferation.

Breast cancer subtypes such as triple-negative that lack the expression of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 receptor (HER2), remain poorly clinically managed due to a lack of therapeutic targets. This necessitates identification and validation of novel targets. Suppression of Popeye domain-containing protein 1 (POPDC1) is known to promote tumorigenesis and correlate to poor clinical outcomes in various cancers, and also promotes cardiac and skeletal muscle pathologies. It remains to be established whether POPDC1 is dysregulated in breast cancer, and whether overcoming the dysregulation of POPDC1 could present a potential therapeutic strategy to inhibit breast tumorigenesis. We assessed the potential of POPDC1 as a novel target for inhibiting breast cancer cell migration and proliferation. POPDC1 was significantly suppressed with reduced cell membrane localization in breast cancer cells. Furthermore, functional suppression of POPDC1 promoted breast cancer cell migration and proliferation, which were inhibited by POPDC1 overexpression. Finally, cAMP interacts with POPDC1 and up-regulates its expression in breast cancer cells. These findings suggest that POPDC1 plays a role in breast tumorigenesis and represents a potential therapeutic target or biomarker in breast cancer medicine.

The a3 isoform of subunit a of the vacuolar ATPase localizes to the plasma membrane of invasive breast tumor cells and is overexpressed in human breast cancer.

The vacuolar (H+)-ATPases (V-ATPases) are a family of ATP-driven proton pumps that acidify intracellular compartments and transport protons across the plasma membrane. Previous work has demonstrated that plasma membrane V-ATPases are important for breast cancer invasion in vitro and that the V-ATPase subunit a isoform a3 is upregulated in and critical for MDA-MB231 and MCF10CA1a breast cancer cell invasion. It has been proposed that subunit a3 is present on the plasma membrane of invasive breast cancer cells and is overexpressed in human breast cancer. To test this, we used an a3-specific antibody to assess localization in breast cancer cells. Subunit a3 localizes to the leading edge of migrating breast cancer cells, but not the plasma membrane of normal breast epithelial cells. Furthermore, invasive breast cancer cells express a3 throughout all intracellular compartments tested, including endosomes, the Golgi, and lysosomes. Moreover, subunit a3 knockdown in MB231 breast cancer cells reduces in vitro migration. This reduction is not enhanced upon addition of a V-ATPase inhibitor, suggesting that a3-containing V-ATPases are critical for breast cancer migration. Finally, we have tested a3 expression in human breast cancer tissue and mRNA prepared from normal and cancerous breast tissue. a3 mRNA was upregulated 2.5-47 fold in all breast tumor cDNA samples tested relative to normal tissue, with expression generally correlated to cancer stage. Furthermore, a3 protein expression was increased in invasive breast cancer tissue relative to noninvasive cancer and normal breast tissue. These studies suggest that subunit a3 plays an important role in invasive human breast cancer.

Protein Subcellular localization profiling of Prostate Cells by Dissociable Antibody MicroArray (DAMA) Staining Technology

Background: Dissociable Antibody MicroArray (DAMA) staining technology is a new approach for global analysis of protein expression and subcellular localization profiles in fixed cells. We have developed the technology and demonstrated its application on identifying differentially expressed proteins in breast cancer cells. We have determined the subcellular localization profiles of 360 proteins in breast cells and identified a protein with unique subcellular localization in breast cancer cells, by combining data from four 96-antibody format arrays. In this report, we have further developed the technology to determine the subcellular localization profiles of 400 proteins simultaneously from single chip, in a 400-antibody format. Method: We have determined the subcellular localization profiles of 400 arrayed antibodies in two normal prostate cell lines (PWR-1E and PZ-HPV-7), and three cancer cell lines (Du145, LNCap and VCap). The subcellular localization profiles were compared and analyzed by Chipview, the program for image database management and analysis. Results: A protein, GRK2 was identified to have unique localization in some of the cancer cells: localized in the membrane of two cancer cell lines, and in the cytosol of normal cell lines and one cancer cell line. The identified subcellular localization difference was confirmed by individual immunostaining. Conclusion: DAMA staining technology could be a powerful method for global subcellular localization profiling in high throughput format.

14-3-3 Proteins interact with FRMD6 and regulate its subcellular localization in breast cancer cells

The Hippo pathway is evolutionarily conserved from Drosophila to mammals. FRMD6 is one crucial upstream component of the Hippo pathway while its function and regulatory mechanism are largely elusive. We decided to purify the protein complex of FRMD6 to further explore its regulatory mechanism. We established the MCF7 breast cancer cells that stably expressed FRMD6 by retroviruses infection. And we purified the FRMD6-interacting protein complex for further analysis by high performance liquid chromatography-mass spectrometry/mass spectrometry(HPLCMS/ MS). Interestingly, we observed that the major binding partner of FRMD6 is 14-3-3 family of proteins. The interaction between FRMD6 and 14-3-3 proteins was detected by co-immunoprecipitation(CO-IP). The disruption of their interaction resulted in the nuclear localization of FRMD6. Importantly, the T28A mutant of FRMD6 showed stronger tumor suppressor function than wild type(WT) FRMD6. Our results indicate that 14-3-3 proteins tightly regulate the subcellular localization of FRMD6 so as to endow FRMD6 with the tumor suppressor function on breast cancer.

Trafficking and Membrane Targeting of NBCn1 in MCF‐7 Breast Cancer Cells

We previously showed that the Na+,HCO3‐ cotransporter NBCn1 (SLC4A7) is upregulated in breast cancer, and that cisplatin treatment of breast cancer cells causes pronounced loss of NBCn1 plasma membrane localization. However, essentially nothing is known regarding the mechanisms of NBCn1 trafficking and turnover. NBCn1 localized predominantly to the basolateral membrane in polarized breast cancer cells, with a highly cell‐density dependent expression profile. Removal of the cytosolic NBCn1 C‐terminal tail (NBCn1‐Ct) strongly reduced NBCn1 membrane localization in HEK293 cells. GST pull‐down in MCF‐7 cells followed by LTQ Orbitrap Mass spectrometry identified Receptor C kinase‐1 (RACK1), retromer complex components vacuolar protein sorting‐associated protein 35 (VPS35) and Sorting Nexin 27 (SNX27), and AP1 and ‐2 adaptor proteins, as NBCn1‐Ct interaction partners. Co‐immunoprecipitation and immunofluorescence analysis confirmed interaction between RACK1 and NBCn1 in MCF‐7 cells. Furthermore, siRNA‐mediated RACK1 knockdown reduced NBCn1 expression and strongly attenuated its plasma membrane localization. SNX27 and VPS35 partially colocalized with NBCn1 in breast cancer cell lines, in a punctate pattern closely adjacent to the plasma membrane, suggestive of a role in NBCn1 trafficking. In conclusion, these preliminary findings point to the involvement of RACK1 and likely also VPS35 and SNX27, in regulation of NBCn1 expression and localization in breast cancer cells.

Mechanobiology of mRNA Localization in Breast Cancer Cells

Introduction: Metastasis occurs when cancer cells form secondary tumors in distant areas of the body. Localization of RNAs in lamellipodial regions has been proposed to play an important role during metastatic progression. In one pathway, the tumor suppressor protein, adenomatous polyposis coli (APC) targets RNAs to cell protrusions. APC -associated mRNAs have been implicated in cellular migration and metastatic progression. Therefore, localization or not of these mRNAs has functional significance in cellular migration and metastasis. Additionally, It has been shown that cancer cells modulate their gene expression in response to the mechanical properties of the substrate. Furthermore, mRNA localization at adhesion sites is influenced by mechanical tension, which is adjusted by cells as a function of the mechanical properties of the cell environment. Therefore, mechanical properties of tissues may play a role during metastasis by modulating localization of mRNAs. As a result, this study investigates APC- associated mRNA localization as a function of substrate stiffness. Methods: We used the MCF10A cell series, a breast cancer progression model composed of cell lines representing pre-malignant to invasive transformation to investigate mRNA localization. By using in situ hybridization to fluorescently label mRNAs, and micropatterned polyacrylamide gels of varying stiffness, we observed APC associated mRNA localization. Glu-tubulin and vinculin were immunostained to study their relationship to mRNA localization. Results: On stiffer substrates (280kPa), we observed increased mRNA localization compared to softer substrates (0.87kPa). Staining of cytoskeletal elements such as Glu-tubulin and vinculin showed a correlation between the abundance and location of the proteins and APC-associated mRNAs. These results reveal biophysical roles of adhesion and cytoskeletal tension in regulating mRNAs to protrusions.

Down regulation of SPAG9 reduces growth and invasive potential of triple-negative breast cancer cells: possible implications in targeted therapy

BackgroundRecently, we reported an association of a novel cancer testis (CT) antigen, sperm-associated antigen 9 (SPAG9) expression in breast cancer clinical samples, indicating its potential role in carcinogenesis. Around 15% breast cancers are designated as triple-negative for which treatment modalities are limited. Therefore, in the present study, we assessed the role of SPAG9 in triple-negative breast cancer cells.MethodsSPAG9 mRNA and protein expression was investigated in various breast cancer cells of different hormone receptor status and different subtypes by employing reverse transcriptase-polymerase chain reaction (RT-PCR), real time PCR, Western blotting, indirect immunofluorescence (IIF) and fluorescence activated cell sorting (FACS). Employing plasmid-based small interfering RNA (siRNA) approach, knockdown of SPAG9 was carried out in triple-negative breast cancer cells, MDA-MB-231, to assess its role on various malignant properties in vitro and in vivo.ResultsSPAG9 mRNA and protein expression was detected in all breast cancer cells. Further, IIF results showed that SPAG9 was predominantly localized in the cytoplasm of breast cancer cells. FACS analysis revealed distinct SPAG9 surface localization in breast cancer cells. Gene silencing of SPAG9 resulted in significant reduction in cellular proliferation, colony forming ability, migration, invasion and cellular motility of MDA-MB-231 cells. Further, ablation of SPAG9 expression resulted in reduction in the tumor growth of human breast cancer xenograft in nude mice in vivo.ConclusionsIn summary, our data indicated that down regulation of SPAG9 reduces growth and invasive potential of triple-negative breast cancer cells, suggesting that SPAG9 may be a potential target for therapeutic use.

A Chimeric p53 Evades Mutant p53 Transdominant Inhibition in Cancer Cells

Because of the dominant negative effect of mutant p53, there has been limited success with wild-type (wt) p53 cancer gene therapy. Therefore, an alternative oligomerization domain for p53 was investigated to enhance the utility of p53 for gene therapy. The tetramerization domain of p53 was substituted with the coiled-coil (CC) domain from Bcr (breakpoint cluster region). Our p53 variant (p53-CC) maintains proper nuclear localization in breast cancer cells detected via fluorescence microscopy and shows a similar expression profile of p53 target genes as wt-p53. Additionally, similar tumor suppressor activities of p53-CC and wt-p53 were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), annexin-V, 7-aminoactinomycin D (7-AAD), and colony-forming assays. Furthermore, p53-CC was found to cause apoptosis in four different cancer cell lines, regardless of endogenous p53 status. Interestingly, the transcriptional activity of p53-CC was higher than wt-p53 in 3 different reporter gene assays. We hypothesized that the higher transcriptional activity of p53-CC over wt-p53 was due to the sequestration of wt-p53 by endogenous mutant p53 found in cancer cells. Co-immunoprecipitation revealed that wt-p53 does indeed interact with endogenous mutant p53 via its tetramerization domain, while p53-CC escapes this interaction. Therefore, we investigated the impact of the presence of a transdominant mutant p53 on tumor suppressor activities of wt-p53 and p53-CC. Overexpression of a potent mutant p53 along with wt-p53 or p53-CC revealed that, unlike wt-p53, p53-CC retains the same level of tumor suppressor activity. Finally, viral transduction of wt-p53 and p53-CC into a breast cancer cell line that harbors a tumor derived transdominant mutant p53 validated that p53-CC indeed evades sequestration and consequent transdominant inhibition by endogenous mutant p53.

Abstract A30: FOXP3 regulates metastatic spread of breast cancer via control of expression of CXCR4 chemokine receptor

Abstract Background: The transcription factor FOXP3 can regulate T cell migration by inhibiting expression of CXCR4, the receptor for the chemokine CXCL12. The increased expression of CXCR4 by breast cancer cells can drive metastatic migration towards sites which express CXCL12. Intracellular trafficking of FOXP3 to the nucleus is required in order for this protein to function. There are three distinct Foxp3 domains that contribute to its nuclear transport. Importantly, human T-cells express at least two splice variants of FOXP3 protein what may affect the structure of nuclear localization regions. We hypothesized that FOXP3 is inactivated in breast cancer due to dysfunctional FOXP3 isoforms causing failure of nuclear localization with subsequent increase in CXCR4 and development of metastasis. Methods: The expression patterns of FOXP3 and CXCR4 were measured at mRNA (qPCR) and protein (immunohistochemistry, immunofluorescence, FACS) levels. 100 sections of benign tissue, cancers, lymph node metastases and 7 breast cell lines were examined with traditional and digital microscopy techniques. The results were correlated with pathological and clinical prognostic indicators of breast cancer. Following comparison of FOXP3 sequences of cancer and normal cells, cancer cells were transfected with FOXP3 vector and changes in their chemotaxis and invasion were investigated. Results: Normal breast epithelial cells (both patient-derived tissues and laboratory cultured cell lines) expressed FOXP3 in their nuclei and at the same time fail to express CXCR4. Breast cancer cells significantly (p<0.05) overexpressed CXCR4, whereas FOXP3 expression was significantly decreased (p<0.05) and confined to the cytoplasm with negligible nuclear expression. Metastases expressed significantly less FOXP3 and more CXCR4 than primaries (p<0.05). We identified a novel FOXP3I12 isoform on cancer cells with insertion of 120bp within first domain of nuclear localization. Transfection of cancer cells with normal FOXP3 plasmid restored physiological nuclear FOXP3 expression. Furthermore, experiments performed with CXCR4-expressing MDA-MB-231 breast cancer cells showed that FOXP3-transfection of this cells effectively inhibited the chemotactic response within a CXCL12 diffusion gradient. Discussion: We demonstrated failure of FOXP3 nuclear localization in breast cancer cells and an inverse correlation between this failure and CXCR4 expression. Isoform FOXP3I12 accumulates outside of the nucleus where it cannot maintain its physiological properties what may lead to increased CXCR4 expression and acquisition of the capacity for the cancer cells to metastasize. Normal FOXP3 reverses its malignant expression pattern and inhibits migration of cancer cells towards their metastatic sites expressing CXCR4. Conclusion: Our findings suggest disruption of FOXP3 nuclear localization due to the dysfunctional isoform FOXP3I12 what may be a novel mechanism of breast cancer invasion and metastases development. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A30.