Metastatic Breast Cancer Tissues Research Articles

Relationship between moesin expression and breast cancer metastasis

Objective To explore the internal relationship between moesin expression and breast cancer metastasis. Methods Immunohistochemistry(IHC),RT-PCR and Western blot were`used to identify moesin expression in breast cancer tissue samples and a series of human breast cancer cell lines with different metastatic potentials.RNAi technique combined with invasion and motility assay in vitro were also used to reveal the interrelationship between moesin deficiency and invasion and motility ability of breast cancer cells.Results IHC analysis showed that moesin were mainly distributed on the cell membrane and cytoplasm.Its expression was higher in metastatic breast cancer tissue samples than in non-metastatic ones(P=0.001,χ2=17.536).Western blot results revealed positive correlation between moesin expression and metastatic potential of breast cancer cells.The results of Transwell experiment displayed that moesin expression silencing remarkably inhibited invasion and motility ability of breast cancer cells,especially the cells with high metastatic potential.After moesin expression,the levels of MTDH mRNA and nm-23mRNA in breast cancer cells(P0.05)significantly decreased,while vimentin expression increased,and the expressions of E-cadherin and MMP-9decreased. Conclusions Moesin over-expression presents a positive correlation to breast cancer metastatic potential.Moesin may participate in the process of invasion and metastasis of breast cancer cells through influencing MTDH and nm-23,enhancing epithelial mesenchymal transition and increasing MMP-9secretion.

Protocadherin-7 induces bone metastasis of breast cancer

Breast cancer had a propensity to metastasize to bone, resulting in serious skeletal complications associated with poor outcome. Previous study showed that Protocadherin-7 (PCDH7) play an important role in brain metastatic breast cancer, however, the role of PCDH7 in bone metastatic breast cancer has never been explored. In the present study, we found that PCDH7 expression was up-regulation in bone metastatic breast cancer tissues by real-time PCR and immunohistochemistry assays. Furthermore, suppression of PCDH7 inhibits breast cancer cell proliferation, migration, and invasion in vitro by MTT, scratch, and transwell assays. Most importantly, overexpression of PCDH7 promotes breast cancer cell proliferation and invasion in vitro, and formation of bone metastasis in vivo. These data provide an important insight into the role of PCDH7 in bone metastasis of breast cancer.

Abstract 514: Targeting of Voltage-gated sodium channel NaV1.5 inhibits cell proliferation and colony formation in breast and ovarian cancer cells.

Abstract Breast cancer is the most common and the second leading cause of death in women worldwide while ovarian cancer is the leading cause of gynecological cancers. Breast and ovarian cancer cells have increased proliferation, invasion/metastasis and resistance to programmed cell death due to mutations, altered signaling pathways and deregulated of control mechanism. One of the differences between normal and cancer cells is the changes in the regulation and the function of ion channels. Ion channels are important signaling molecules expressed in a wide range of tissues where they have significant involvement in determining a variety of cellular functions: solute transport, volume control, enzyme activity, secretion, invasion, gene expression, excitation-contraction coupling and intercellular communication. During the malignant transformation, a series of genetic alterations occur, which may also affect the expression and activity of ion channels. This abnormal ion channel activity has been hypothesized to support cell proliferation and tumor growth. Voltage-gated sodium channels (VGNaC) are classically described as critical elements of action potential initiation and propagation in excitable cells. Recent studies showed that VGNaC expression was significantly up-regulated in metastatic human breast cancer cells and tissues, and VGNaC activity potentiated cellular directional motility, endocytosis and invasion. However, the mechanisms of the VGNaC and its functional relevance to breast cancer proliferation are currently not well-known. In this study, we investigated that the effects of downregulation NaV1.5 channel expression by specific RNA interference (ie siRNA) in breast and ovarian cancer cells in vitro. In our work, we first determined the effects of Adult NaV1.5 siRNA and analyzed the effects of Adult and Neonatal (neonatal splice variant of NaV1.5) NaV1.5 silencing by siRNA in BCa cells by Real-Time PCR. Our Real-Time PCR results showed that Adult NaV1.5 channel siRNA reduced the level of NaV1.5 mRNA for MDA-MB-231 BCa cells. We found that knockdown of NaV1.5 channel by siRNA significantly inhibited MDA-MB-231 breast and SKOV3-IP1, SKOV3-TR and HEYA8 ovarian cancer cell proliferation and colony formation. We are currently investigating downstream cellular pathways involved in NaV1.5 mediated proliferation and other cellular functions. In conclusion, our results demonstrate that targeting of NaV1.5 in breast and ovarian cancer cells decreases cell proliferation and colony formation and it may be used as a new therapeutic target in these cancers. Citation Format: Mumin A. Erdogan, Bulent Ozpolat. Targeting of Voltage-gated sodium channel NaV1.5 inhibits cell proliferation and colony formation in breast and ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 514. doi:10.1158/1538-7445.AM2013-514

Post-translational Modifications of Recombinant Human Lysyl Oxidase-like 2 (rhLOXL2) Secreted from Drosophila S2 Cells*

Human lysyl oxidase-like 2 (hLOXL2) is highly up-regulated in metastatic breast cancer cells and tissues and induces epithelial-to-mesenchymal transition, the first step of metastasis/invasion. hloxl2 encodes four N-terminal scavenger receptor cysteine-rich domains and the highly conserved C-terminal lysyl oxidase (LOX) catalytic domain. Here, we assessed the extent of the post-translational modifications of hLOXL2 using truncated recombinant proteins produced in Drosophila S2 cells. The recombinant proteins are soluble, in contrast to LOX, which is consistently reported to require 2-6 m urea for solubilization. The recombinant proteins also show activity in tropoelastin oxidation. After phenylhydrazine derivatization and trypsin digestion, we used mass spectrometry to identify peptides containing the derivatized lysine tyrosylquinone cross-link at Lys-653 and Tyr-689, as well as N-linked glycans at Asn-455 and Asn-644. Disruption of N-glycosylation by site-directed mutagenesis or tunicamycin treatment completely inhibited secretion so that only small quantities of inclusion bodies were detected. The N-glycosylation site at Asn-644 in the LOX catalytic domain is not conserved in human LOX (hLOX), although the LOX catalytic domain of hLOX shares ∼50% identity and ∼70% homology with hLOXL2. The catalytic domain of hLOX was not secreted from S2 cells using the same expression system. These results suggest that the N-glycan at Asn-644 of hLOXL2 enhances the solubility and stability of the LOX catalytic domain.

Abstract P2-10-16: Quantitative HER3 protein expression and PIK3CA mutation status in matched samples from primary and metastatic breast cancer tissues and correlation with time to recurrence

Abstract Background: HER3 is thought to play a prominent role in resistance to HER2-directed breast cancer therapies. Recent data suggest that HER3 levels also influence HER2-normal breast tumor biology. HER3 and PI3K signaling are linked in that in HER3 signaling activates PI3K and inhibition of PI3K activity can upregulate HER3 expression. Here, we measured quantitative HER3 protein expression levels and PIK3CA mutation status in matched tissues from the primary tumor and site of metastasis to assess correlations with time to recurrence. Methods: 44 pairs (8 HER2+ by HERmark®) of matched tissues from the primary tumor and the site of metastasis were evaluated for HER3 protein expression using a sensitive, quantitative assay for HER3 protein expression in FFPE tissue sections (VeraTag®). Matched samples were also evaluated for quantitative HER2 expression (HERmark) and for PIK3CA mutations at exon 9 (E542K and E545K) and exon 20 (H1047R). Results: HER3 protein expression at the metastatic site was largely independent of HER3 levels at the primary site (Spearman p = 0.50) in contrast to HER2 expression (Spearman p = 0.0004). HER3 expression in the primary tumor correlated with time to recurrence (TTR) (HR = 2.0 per 2-fold increase in HER3; p < 0.0001). Conversely, HER3 expression measured at the site of metastasis was not correlated with TTR (p = 0.55). Estrogen receptor negative tumors were less likely to have PIK3CA mutations (p = 0.023). In cases of primary tumors with PIK3CA mutations, no reversions to wild-type PIK3CA were observed in the metastatic sites. In metastatic tumors, mutations detected in the primary tumor as well as new mutations were observed. A gain of an exon 9 mutation at the metastatic site correlated with shorter TTR (HR = 2.5; p = 0.043). Excluding the 8 samples that were HER2+ by HERmark, longer TTR was observed for patients with PIK3CA mutations in the primary tumor (HR = 0.47; p = 0.042), which is consistent with previous reports. Interestingly, the longer TTR for those with PIK3CA mutations appeared to be dependent on quantitative HER3 protein level (interaction p = 0.065). Conclusions: HER3 protein expression in matched primary and metastatic breast cancer tissues were unrelated. This may indicate that HER3 protein is influenced by the different tumor microenvironments of the primary and metastatic sites. PIK3CA mutations were either maintained or acquired at metastatic sites. Both low HER3 protein expression and the presence of PIK3CA mutations in the primary tumor but not the metastatic tumor were associated with longer TTR. These observations suggest that HER3 protein expression may be an important prognostic factor for breast cancer progression. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-16.

Abstract P4-08-08: HOXC10, a homeobox protein overexpressed in breast cancer, modulates the response to chemotherapy treatment.

Abstract Background: Breast cancer is the second leading cause of cancer deaths in women worldwide. Although chemotherapy is effective, resistance to drugs develops over time and can account for treatment failure in over 90% of metastatic breast cancer patients. HOX genes are homeobox-containing transcription factors well-known for their role in morphogenesis. and during carcinogenesis and metastasis. The goal of this study is to understand the role of HOXC10 in breast cancer and mainly in response to chemotherapy. Methods: Using a tiling array of all four HOX clusters in a panel of primary and metastatic breast cancer tissues, we identified HOXC10 as being among the highly overexpressed genes in breast cancer. Then using a panel of cell lines that either stably overexpress exogenous HOXC10 or cell lines with stably downregulated endogenous HOXC10 (mediated by shRNA), we investigated the role of HOXC10 in proliferation, response to chemotherapy treatment and repair of DNA damage. Results: HOXC10 is overexpressed in 67% of primary breast tumors (n = 31), in 82% of the metastatic tissues (n = 49) and in most breast cancer cell lines (n = 48). In vitro and in vivo investigation confirmed that HOXC10 plays an oncogenic role in breast cancer. Further, knockdown of HOXC10 in a panel of breast cancer cell lines slowed their proliferation and arrested them at the G1 phase, by inactivating the RB/E2F pathway, decreasing the number of new origins and eventually reducing the polyploidy population. Cell survival assays after different chemotherapeutic drug treatment showed that overexpression of the exogenous HOXC10 in MCF10A led to less susceptibility to most drugs. This was partially due to a protection from apoptosis by upregulating and activating the anti-apoptotic machinery such as the NF-kb pathway. Further investigation revealed the involvement of HOXC10 in DNA repair (and not initial response), especially after DNA crosslink damage. Interestingly, the binding of HOXC10 to CDK7 in a region outside its homeodomain activates CDK7 activity towards RNA polymerase II mainly in response to DNA damage. Since HOX genes are difficult to target therapeutically, one potential approach to overcome chemoresistance in HOXC10 overexpressing cells is by including CDK7 inhibitors (already in clinical trials). All these results were confirmed in the SUM159 model which stably expresses a HOXC10-shRNA. Finally, HOXC10 was found to be significantly overexpressed in MCF7 isogenic cell lines gradually selected to be resistant to some chemotherapeutic drugs. By knocking down HOXC10 in these sublines, resistance to the drug was reduced. Further, SUM159 and MDAMD231 xenografts that were treated with chemotherapy over weeks and that show partial to no response tend to have a higher expression of HOXC10. Conclusion: This study shows that HOXC10, a homeobox protein previously shown to be regulated during the cell cycle and to have a positive effect on proliferation, is overexpressed in the majority of breast cancers. This upregulation may have clinical implications since cells with higher expression of HOXC10 tend to have more genomic instability and activation of anti-apoptotic and DNA repair pathways, which eventually modulate the response to some chemotherapy drugs. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-08-08.

S100A4 promotes invasion and angiogenesis in breast cancer MDA-MB-231 cells by upregulating matrix metalloproteinase-13.

S100A4 is a member of the S100 family of calcium-binding proteins that is directly involved in tumor metastasis. In the present study, we examined the potential role of S100A4 in metastasis in breast cancer and its relation with matrix metalloproteinase-13 (MMP-13). Analysis of 100 breast cancer specimens including 50 with and 50 without lymph node metastasis showed a significant upregulation of S100A4 and MMP-13 expression in metastatic breast cancer tissues. Positive immunoreactivity for S100A4 was associated with MMP-13 expression. Overexpression of S100A4 in the MDA-MB-231 breast cancer cell line upregulated MMP13 expression leading to increased cell migration and angiogenesis. SiRNA-mediated silencing of S100A4 downregulated MMP13 expression and suppressed cell migration and angiogenesis. Moreover, neutralization of MMP-13 activity with a specific antibody blocked cell migration and angiogenesis in MDA-MB-231/S100A4 cells. In vivo siRNA silencing of S100A4 significantly inhibited lung metastasis in transgenic mice. The present results suggest that the S100A4 gene may control the invasive potential of human breast cancer cells by modulating MMP-13 levels, thus regulating metastasis and angiogenesis in breast tumors. S100A4 could therefore be of value as a biomarker of breast cancer progression and a novel therapeutic target for human breast cancer treatment.

MicroRNA-34 suppresses breast cancer invasion and metastasis by directly targeting Fra-1

MicroRNAs have key roles in tumor metastasis. Here, we describe the regulation and function of miR-34a and miR-34c (miR-34a/c) in breast cancer metastasis. Expression analysis verified that miR-34a/c expression is significantly decreased in metastatic breast cancer cells and human primary breast tumors with lymph node metastases. Overexpression of miR-34a/c could inhibit breast cancer cell migration and invasion in vitro and distal pulmonary metastasis in vivo. Further studies revealed that Fos-related antigen 1 (Fra-1 or Fosl1) is a downstream target of miR-34a/c as miR-34a/c bound directly to the 3'untranslated region of Fra-1, subsequently reducing both the mRNA and protein levels of Fra-1. Silencing of Fra-1 recapitulated the effects of miR-34a/c overexpression, whereas enforced expression of Fra-1 reverses the suppressive effects of miR-34a/c. Moreover, significant downregulation of miR-34a in metastatic breast cancer tissues was found to be inversely correlated with Fra-1 expression. Our results demonstrate that miR-34a/c functions as a metastasis suppressor to regulate breast cancer migration and invasion through targeting Fra-1 oncogene and suggest a therapeutic application of miR-34 in breast cancer.

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.

Quantitative HER2 measurement and PI3K mutation profile in matched primary and metastatic breast cancer tissues.

614 Background: HER2 status of primary breast cancer (PBC) is routinely used to determine systemic treatment for metastatic breast cancer (MBC) patients. Discordance rates of HER2 status between PBC and MBC range from 5.5% to 29% based on published meta-analyses. The clinical benefit of re-assessing HER2 in MBC tissues remains controversial. In this study, we measured quantitative HER2 expression in matched PBC and MBC tissues and correlated changes of HER2 with mutations in the catalytic domain of PI3 kinase(PIK3CA). Methods: Total HER2 protein expression (H2T) was quantified by the HERmark assay in 41 matched PBC and MBC formalin-fixed, paraffin-embedded specimens. PIK3CA mutation status in exons 9 (E545K and E542K) and 20 (H1047R) was determined using a validated pyrosequencing assay. Results: MBC samples included 5 lymph node, 13 viscera, 6 brain, and 17 soft tissue lesions (N=41). 27 (66%) cases showed higher H2T in MBC than in matched PBC; and 14 (34%) cases had higher H2T in PBC than in matched MBC, indicating an overall increase of H2T in matched MBC lesions (fold change 0.25-17.57; p=0.005, paired Wilcoxon rank sum test). HER2 positive conversion (HERmark negative/equivocal in PBC, but positive in matched MBC) was found in 6 (15%) cases, while HER2 negative conversion (HERmark positive in PBC, but negative/equivocal in matched MBC) was seen in 2 (5%) cases. HER2 status was unchanged in 33 (80%) cases. PIK3CA mutations were detected in 13 (32%) of PBC and 19 (46%) of MBC samples. Among the HER2 positive conversion cases, PIK3CA mutation was identified in 50% (3/6) PBC and 67% (4/6) MBC, compared to 0% (0/2, PBC or MBC) in the HER2 negative conversion cases. Among cases with unchanged HER2 status, PIK3CA mutation was observed in 30% (10/33) PBC and 42% (14/33) MBC. Conclusions: Quantitative HER2 assessment revealed a 20% discordance in HER2 status between matched PBC and MBC tissues, with more frequent conversion from low HER2 in PBC to high HER2 in MBC. PIK3CA mutation was observed more frequently in patients who converted from HER2 negative PBC to HER2 positive MBC. These results suggest that re-assessment of biomarkers in MBC tissues may better inform the selection of therapeutic options for patients with MBC.

Abstract 694: Characterization of a novel monoclonal antibody to Glia maturation factor-beta showing significant clinical utility in the identification of breast carcinoma

Abstract Background: Glia maturation factor beta (GMF-beta) is a growth and differentiation factor of brain cells, stimulation of neural degeneration and inhibition of proliferation of tumor cells. It was recently found to involve in the progression of serous ovarian carcinoma. In the present study, we generated a novel antibody against human GMF-beta and investigated the potential of GMF-beta as a biomarker in breast cancer. Methods and findings: We generated a novel monoclonal antibody (mAb), AB-GMF-beta using hybridoma technology and native protein as an immunogen. This mAb is of IgG1 (kappa) type. It specifically recognized both native and denatured human GMF-beta protein and displayed high affinity as assessed by ELISA, immunohistochemistry assay, western blot and flow cytometry applications. Using this mAb in competitive ELISA, we showed for the first time that soluble GMF-beta level is significantly increased in metastatic breast cancer patients’ plasma which directly correlated with GMF-beta expression pattern in metastatic breast cancer tissues. Tissue expression of GMF-beta was determined by using immunohistochemical techniques. Seventy normal and 241 breast cancer tissues were included which consist of 194 ductal and 47 lobular carcinoma samples. Most of normal tissues showed negative or weak nuclear staining, while different levels of nuclear, and diffuse and weak cytoplasmic staining were observed in cancer sections. To evaluate the immunoreactivity of GMF-beta associated with tumor histological types, the staining intensity in nucleus were scored as 0, 1+, 2+ and 3+. Grade 0 and 1+ were defined as negative and 2+ and 3+ as positive. Forty four percent of ductal carcinomas showed strong staining whereas it is significantly increased to 72.3% in lobular tumors (p<0.001). Conclusion: The novel AB-GMF-beta mAb showed high performance in blood and tissue assays. The circulating GMF-beta levels and expressions in breast tissues are significantly increased in breast cancer patients in comparison of normal controls. The increased tissue expressions in breast carcinoma associate with histological tumor types. Our data suggest the potential clinical use of GMF-beta as a biomarker for diagnosis and prognosis in blood and tissue as well as a therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 694. doi:1538-7445.AM2012-694

Abstract 2297: MicroRNAs inhibit breast canncer metastasis via targeting CXC chemokine receptor 4

Abstract Overexpressed CXC chemokine receptor (CXCR4) strongly contributes to breast cancer metastasis. However, the underlying mechanisms of CXCR4 overexpression need still to be explored. MicroRNAs (miRNA) have been implicated as a class of critical regulators controlling diverse biological processes. In the present study, we investigated whether miR-302 and miR-588 are involved in the regulation of CXCR4 and play a functional role in breast cancer metastasis. We have shown that miR-302 and miR-588 were downregulated in highly metastatic breast cancer cell lines and tissue specimens compared to low metastatic cell lines and breast cancer tissues. The decreased expression levels were inversely consistent with overexpression of CXCR4 in highly breast cancer cell lines and tissues. In addition, we found that restoration of miR-302 and miR-588 resulted in a dramatic reduction of CXCR4 expression at protein levels by directly targeting its 3′ untranslated region. Furthermore, the elevated expression of miR-302 and miR-588 significantly inhibited the invasion of breast cancer cells. More promisingly, our data have demonstrated that overexpression of miR-302 reduced metastasis and growth of breast cancer in vivo. Our findings describe a new mechanism for the regulation of CXCR4 and metastasis of breast cancer. The newly characterized miR-302/588/CXCR4 link provides potential molecular markers for predicting breast cancer metastasis. MiR-302 and miR-588 may have a therapeutic potential to suppress breast cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2297. doi:1538-7445.AM2012-2297

Abstract 695: Significant loss of nuclear expression of Actinin-4 in metastatic breast carcinoma and lymph nodes: A novel biomarker for metastatic breast carcinoma

Abstract Background: Actinin-4 is an actin cross-linked protein. The expression and subcellular distribution of actinin-4 (ACTN4) has been associated with multiple roles in tumorigenicity and cancer metastasis. In the present study, we developed a novel antibody against human actinin-4 and investigated the clinical utility of actinin-4 as a biomarker in metastatic breast cancer. Methods and findings: We generated a novel monoclonal antibody (AB-Actinin-4) using hybridoma technology and native ACTN4 protein as an immunogen. The antibody specifically recognized both native and denatured human Actinin-4 protein and displayed high affinity as assessed by ELISA, immunohistochemistry assay, western blot and flow cytometry. Using this antibody and ELISA as well as immunohistochemical techniques, we examined plasma levels and tissue expression of actinin-4 in healthy controls’ and breast cancer patients’ samples. 50 plasma samples from healthy controls and breast cancer patients were analyzed for the presence of soluble actinin-4. ELISA results showed significantly decreased actinin-4 levels in plasma samples from patients with breast carcinomas compared to healthy control plasma. A total of 156 cases, including 70 normal, 36 non-metastatic breast carcinoma, 50 breast carcinoma tissues with regional metastasis, and 50 matched metastatic lymph nodes were examined. The immunoreactivity of actinin-4 was located mainly in nucleus and cytoplasm of cells. To assess the difference between groups, the staining intensity and intracellular distribution were compared and the intensity in nucleus and cytoplasm was separately scored as 0, 1+, 2+ and 3+. Grades 0 and 1+ were defined as negative and 2+ and 3+ as positive. Significant differences between the groups were found in nuclear staining. 92% of normal and 89% of non-metastatic breast carcinoma samples displayed positive nuclear staining. In contrast, only 29% of metastatic lymph nodes and 51% of metastatic breast cancer tissues showed strong nuclear staining. Conclusion: Actinin-4 demonstrated significantly decreased expression in plasma from breast cancer patients. Metastasis leads to significant loss of the nuclear expression of Actinin-4 in breast cancer tissues, in comparison to normal and non-metastatic breast carcinoma tissues. Our data suggests that Actinin-4 might have significant impact in breast cancer metastasis. Actinin-4 may serve as a reliable biomarker for the diagnosis and prognosis of metastatic breast cancer as well as could be a therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 695. doi:1538-7445.AM2012-695

Intracellular and extracellular human lysyl oxidase‐like 2 in metastatic breast cancer cells

Human lysyl oxidase‐like 2 (HLOXL2) is highly up‐regulated in metastatic breast cancer cells and tissues. Native and recombinant HLOXL2s (rHLOXL2s) are soluble and mostly secreted into the media, but also found in cytosol and nucleus. Secreted HLOXL2 (~100 kDa) was confirmed for the presence of glycosylation, proteolysis, and formation of lysine tyrosylquinone cofactor. The fourth SRCR and catalytic domains of HLOXL2 are in communication to achieve optimal activity, and N‐glycosylation at Asn455 and Asn644 is essential for protein secretion. MCF‐7 cells stably expressing rHLOXL2s show higher proliferation and invasiveness in vitro, but do not exhibit characteristic metastasis molecular markers. Addition of β‐Aminopropionitrile, the specific inhibitor of HLOXL2, significantly abolished proliferation and invasiveness, as in the case for MDA‐MB‐231. Intracellular HLOXL2 (~80 kDa) is unglycosylated, but catalytically competent. Wild type MCF‐7 cells can uptake the purified secreted rHLOXL2s, and the exogenous N‐glycosylated rHLOXL2s localize in the nucleus and induce epithelial‐to‐mesenchymal transition. Evidence supports that HLOXL2 goes beyond its traditional role of initiating collagen and elastin cross‐linking extracellularly, its intracellular accumulation leads to a dramatic increase of metastasis/invasion in breast cancer cells.

Abstract A57: Distinctive phosphoprotein profiles in a benign breast tissue, and triple-negative primary and metastatic breast cancer tissues from the same African American woman by LC-LTQ/FT-ICR mass spectrometry

Abstract African-American women have a higher risk for developing triple-negative breast cancer (TNBC). Lacking the expression of receptors for estrogen and progesterone, and without human epidermal growth factor 2 receptor gene amplification, TNBC is a very aggressive type of breast cancer with a high likelihood of metastasis and recurrence. Specific therapeutic targets for this aggressive disease remain to be identified. Phosphorylation, a post-translational modification that adds one or more phosphate groups to a protein, plays a key role in the activation and deactivation of a protein's cellular function. Here we report the first systematic phosphoproteomic analysis of a benign breast tissue, a primary breast cancer tissue, and a metastatic breast cancer tissue from the same African-American woman. Differential phosphoprotein levels were measured with a reversed-phase nano-liquid chromatograph coupled to a hybrid linear quadrupole ion trap/Fourier transform ion cyclotron resonance mass spectrometer (LC-LTQ/FT-ICR MS). Five proteins are found to be highly phosphorylated in the metastasis site of a triple negative breast cancer patient, e.g. hepatoma-derived growth factor and Isoform HMG-Y of high mobility group protein HMG-I/HMG-Y. Six proteins are found to be highly phosphorylated in the primary cancer site of a triple negative breast cancer patient, e.g. isoform 2 of transcription intermediary factor 1-beta and isoform 1 of clathrin heavy chain 1. Identified phosphoproteins are known to be involved in breast cancer signal transduction pathways. For example, the decrease in sumoylation by doxorubicin of isoform 2 of transcription intermediary factor 1-beta (TRIM28) protein was reported to be essential to induce p21 expression, and thus cell growth inhibition in MCF-7 breast cancer cells. In patients with invasive breast cancers, the co-expression of heat-shock protein (HSP) 90 alpha with an overexpressed kinase, or along with the loss of a phosphatase and tensin homologue deletion on chromosome 10, is a putative molecular prognostic marker to predict early relapse. Moreover, HSP90-alpha, which is expressed extracellularly from breast cancer cells, interacts with matrix metalloproteinase-2 (MMP2). This interaction promotes MMP2 activation, which is crucial for tumor invasiveness. HSP90-alpha is also involved in several signal pathways related to breast cancer. These results may identify new diagnostic biomarkers and therapeutic targets for TNBC. (This work was supported by grant BCTR0504465 from the Susan G. Komen for the Cure Breast Cancer Foundation, grants from the Florida Breast Cancer Coalition Research Foundation, and the Florida State University to Prof. Q.-X. Sang and by NSF Division of Materials Research through DMR-0654118 and the State of Florida to Prof. Alan G. Marshall. We also acknowledge the Cooperative Human Tissue Network Southern Division, University of Alabama at Birmingham, from which we obtained the tissues for this project.) Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):A57.

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.

Differential phosphopeptide expression in a benign breast tissue, and triple-negative primary and metastatic breast cancer tissues from the same African-American woman by LC-LTQ/FT-ICR mass spectrometry

African-American women have a higher risk for developing triple-negative breast cancer (TNBC). Lacking the expression of receptors for estrogen and progesterone, and without human epidermal growth factor 2 receptor gene amplification, TNBC is a very aggressive type of breast cancer with a high likelihood of metastasis and recurrence. Specific therapeutic targets for this aggressive disease remain to be identified. Phosphorylation, a post-translational modification that adds one or more phosphate groups to a protein, plays a key role in the activation and deactivation of a protein’s cellular function. Here, we report the first systematic phosphoproteomic analysis of a benign breast tissue, a primary breast cancer tissue, and a metastatic breast cancer tissue from the same African-American woman. Differential phosphoprotein levels were measured with reversed-phase nano-liquid chromatography coupled to a hybrid linear quadrupole ion trap/Fourier transform ion cyclotron resonance mass spectrometer (LC-LTQ/FT-ICR MS). Five proteins were found to be highly phosphorylated in the metastatic site whereas six proteins were highly phosphorylated in the cancer site of the TNBC patient. Identified phosphoproteins are known to be involved in breast cancer signal transduction pathways and these results may identify new diagnostic and therapeutic targets for TNBC.

Abstract 4564: huHER3-8, a novel humanized anti-HER3 antibody that inhibits exogeneous ligand-independent proliferation of tumor cells

Abstract HER3 (ErbB3) is a member of the human epidermal growth factor receptor (HER) tyrosine kinase family that includes EGFR and HER2, two well-known targets for oncology therapy. Kinase signaling of the HER family is initiated by ligand binding that induces receptor homodimerization or heterodimerization and transphosphorylation of the intracellular domain. In this regards, HER3 is unique among the HER kinase family members. Despite being able to bind ligand, HER3 does not possess a catalytically active kinase domain. To turn on the signaling pathway, HER3 needs to form a heterodimer with another HER family member. However, this does not make HER3 inferior with respect to HER pathway signaling, since dimerization of HER3 with HER2 elicits one of the most powerful oncogenic signals. Similar to EGFR and HER2, HER3 expression and activity have been linked to the pathogenesis of various cancers including breast, ovarian and melanoma, and has been associated with resistance to several cancer therapies. The significance of HER3 in cancer pathogenesis prompted us to develop HER3 antagonistic antibodies. A large number of HER3 antibodies were generated by immunizing mice with HER3 expressing cell lines, and screened for the ability to bind human and monkey HER3. Additionally, since HER3 and activated HER3 (phospho-HER3) were detected in the majority of primary as well as metastatic breast cancer tissues by immunohistochemistry staining, antibodies were screened for their ability to inhibit the HER3 ligand-induced proliferation as well as the basal proliferation of breast cancer cell lines. Three HER3 antibodies, HER3-3, HER3-8 and HER3-10, were exceptionally potent in inhibiting the basal proliferation of four breast cancer cell lines that constitutively express phospho-HER3, where other HER3 antibodies (i.e. U1-59 and MM-121) had little or no activity. In SKBR3 cells, these HER3 antibodies inhibited cell proliferation, comparable to the activity of trastuzumab although the HER3 expression level was thirty-fold lower than the HER2 level. All three HER3 antibodies recognized overlapping epitopes. However, only HER3-8 and HER3-10 antibodies strongly competed for the binding of HER3 ligand and inhibited HER2-HER3 dimerization. These HER3 antibodies were also very potent in inhibiting ligand-induced HER3 signaling and tumor cell proliferation. Because of its best overall activities, HER3-8 was humanized by variable domain resurfacing technology. Humanized HER3-8 retained all of the biological properties and activities of muHER3-8. In conclusion, we have generated a novel humanized HER3 antibody, huHER3-8 that is not only active in inhibiting exogenous ligand-induced HER3 signaling and tumor cell growth, but also effective in inhibiting the basal proliferation of HER3-positive tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4564. doi:10.1158/1538-7445.AM2011-4564

P183 Prognostic impact of discordance of biological markers between primary and metastatic breast cancer tissue from autopsy

P183 Prognostic impact of discordance of biological markers between primary and metastatic breast cancer tissue from autopsy

Abstract P4-01-03: The Homeobox Protein H0XC10 Is Overexpressed in Breast Cancer and Confers Resistance to Chemotherapy

Abstract Background: Breast cancer is the second leading cause of cancer deaths in women worldwide. Although chemotherapy is effective, resistance to drugs develops over time and can account for treatment failure in over 90% of metastatic breast cancer patients. HOX genes are homeobox-containing transcription factors well-known for their role in morphogenesis. However, accumulating evidence has emphasized their importance during carcinogenesis and metastasis. The goal of this study is to understand the role of HOXC10 in breast cancer and the consequence of its overexpression in conferring chemotherapy resistance. Methods: We conducted a tiling array of all four HOX clusters to identify dysregulated HOX genes in a panel of primary and metastatic breast cancer tissues, and validated the overexpression of HOXC10 in metastatic breast cancer. Next we established cell lines that stably overexpress HOXC10 and others where endogenously overexpressed HOXC10 was silenced by shRNAs, and determined phenotypic and biochemical changes resulting from these manipulations. Results: HOXC10 is overexpressed in 67% of primary breast tumors (n=31), in 82% of the metastatic tissues (n=49) and in most breast cancer cell lines. When compared to cancers at other sites, overexpression tends to be significantly higher in breast cancer, and correlates with increasing grade and tumor size. Cell survival assays (MTT and colony formation) after drug treatment show that overexpression of exogenous HOXC10 in MCF10A conferred drug resistance. Further molecular examination revealed that overexpression of HOXC10 led to a dysregulation in the Rb-E2F pathway, and therefore the G1/S checkpoint was affected. In addition, the tendency for the formation of 8N cells and the concomitant protection from apoptosis suggested that HOXC10 overexpression might lead to genomic endoreduplication and instability. This fact along with the strikingenhanced recovery of the cells from pactitaxel treatment (as compared to the recovery from any other chemotherapeutic drug) and the protection from mitotic catastrophe implied a role of HOXC10 in the mitotic checkpoint. This was confirmed by a dysregulation of the expression of the genes involved in this checkpoint. All these experiments were further validated by stably expressing a shRNA to HOXC10 in SUM159 breast cancer cells with high endogenous HOXC10 levels. Conclusion: Our studies show for the first time that HOXC10, a homeobox protein previously shown to be regulated during the cell cycle and to have a positive effect on proliferation, is overexpressed in the majority of breast cancers. This upregulation may have some clinical implications since cells with higher expression of HOXC10 tend to have more invasive properties, more genomic instability, and are more resistant to some chemotherapy drugs. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-01-03.