G protein-coupled kisspeptin receptor induces metabolic reprograming and tumorigenesis in estrogen receptor-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly metastatic and deadly disease. TNBC tumors lack estrogen receptor (ERα), progesterone receptor (PR), and HER2 (ErbB2) and exhibit increased glutamine metabolism, a requirement for tumor growth. The G protein-coupled kisspeptin receptor (KISS1R) is highly expressed in patient TNBC tumors and promotes malignant transformation of breast epithelial cells. This study found that TNBC patients displayed elevated plasma kisspeptin levels compared with healthy subjects. It also provides the first evidence that in addition to promoting tumor growth and metastasis in vivo, KISS1R-induced glutamine dependence of tumors. In addition, tracer-based metabolomics analyses revealed that KISS1R promoted glutaminolysis and nucleotide biosynthesis by increasing c-Myc and glutaminase levels, key regulators of glutamine metabolism. Overall, this study establishes KISS1R as a novel regulator of TNBC metabolism and metastasis, suggesting that targeting KISS1R could have therapeutic potential in the treatment of TNBC.

Isoform-specific promotion of breast cancer tumorigenicity by TBX3 involves induction of angiogenesis.

TBX3 is a member of the highly conserved family of T-box transcription factors involved in embryogenesis, organogenesis and tumor progression. While the functional role of TBX3 in tumorigenesis has been widely studied, less is known about the specific functions of the different isoforms (TBX3iso1 and TBX3iso2) which differ in their DNA-binding domain. We therefore sought to investigate the functional consequence of this highly conserved splice event as it relates to TBX3-induced tumorigenesis. By utilizing a nude mouse xenograft model, we have identified differential tumorigenic potential between TBX3 isoforms, with TBX3iso1 overexpression more commonly associated with invasive carcinoma and high tumor vascularity. Transcriptional analysis of signaling pathways altered by TBX3iso1 and TBX3iso2 overexpression revealed significant differences in angiogenesis-related genes. Importantly, osteopontin (OPN), a cancer-associated secreted phosphoprotein, was significantly up-regulated with TBX3iso1 (but not TBX3iso2) overexpression. This pattern was observed across three non/weakly-tumorigenic breast cancer cell lines (21PT, 21NT, and MCF7). Up-regulation of OPN in TBX3iso1 overexpressing cells was associated with induction of hyaluronan synthase 2 (HAS2) expression and increased retention of hyaluronan in pericellular matrices. These transcriptional changes were accompanied by the ability to induce endothelial cell vascular channel formation by conditioned media in vitro, which could be inhibited through addition of an OPN neutralizing antibody. Within the TCGA breast cancer cohort, we identified an 8.1-fold higher TBX3iso1 to TBX3iso2 transcript ratio in tumors relative to control, and this ratio was positively associated with high-tumor grade and an aggressive molecular subtype. Collectively, the described changes involving TBX3iso1-dependent promotion of angiogenesis may thus serve as an adaptive mechanism within breast cancer cells, potentially explaining differences in tumor formation rates between TBX3 isoforms in vivo. This study is the first of its kind to report significant functional differences between the two TBX3 isoforms, both in vitro and in vivo.

TBX3 promotes progression of pre-invasive breast cancer cells by inducing EMT and directly up-regulating SLUG.

The acquisition of cellular invasiveness by breast epithelial cells and subsequent transition from ductal carcinoma in situ (DCIS) to invasive breast cancer is a critical step in breast cancer progression. Little is known about the molecular dynamics governing this transition. We have previously shown that overexpression of the transcriptional regulator TBX3 in DCIS-like cells increases survival, growth, and invasiveness. To explore this mechanism further and assess direct transcriptional targets of TBX3 in a high-resolution, isoform-specific context, we conducted genome-wide chromatin-immunoprecipitation (ChIP) arrays coupled with transcriptomic analysis. We show that TBX3 regulates several epithelial-mesenchymal transition (EMT)-related genes, including SLUG and TWIST1. Importantly, we demonstrate that TBX3 is a direct regulator of SLUG expression, and SLUG expression is required for TBX3-induced migration and invasion. Assessing TBX3 by immunohistochemistry in early-stage (stage 0 and stage I) breast cancers revealed high expression in low-grade lesions. Within a second independent early-stage non-high-grade cohort, we observed an association between TBX3 level in the DCIS and size of the invasive focus. Additionally, there was a positive correlation between TBX3 and SLUG, and TBX3 and TWIST1 in the invasive carcinoma. Pathway analysis revealed altered expression of several proteases and their inhibitors, consistent with the ability to degrade basement membrane in vivo. These findings strongly suggest the involvement of TBX3 in the promotion of invasiveness and progression of early-stage pre-invasive breast cancer to invasive carcinoma through the low-grade molecular pathway. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Invadopodia are chemosensing protrusions that guide cancer cell extravasation to promote brain tropism in metastasis.

Invadopodia are cell protrusions that mediate cancer cell extravasation but the microenvironmental cues and signaling factors that induce invadopodia formation during extravasation remain unclear. Using intravital imaging and loss of function experiments, we determined invadopodia contain receptors involved in chemotaxis, namely GABA receptor and EGFR. These chemotaxis capabilities are mediated in part by PAK1 which controls invadopodia responsiveness to ligands such as GABA and EGF via assembly, stability, and turnover of invadopodia in vivo. PAK1 knockdown rendered cells unresponsive to chemotactic stimuli present in the stroma, resulting in dramatically lower rates of cancer cell extravasation and metastatic colony formation compared to stimulated cancer cells. In an experimental mouse model of brain metastasis, inhibition of PAK1 significantly reduced overall tumor burden and reduced the average size of brain metastases. In summary, invadopodia contain chemotaxis receptors that can respond to microenvironmental cues to guide cancer cell extravasation, and when PAK1 is depleted, brain tropism of metastatic breast cancer cells is significantly reduced, blocking secondary colony growth at sites otherwise permissive for metastatic outgrowth.

The matrix protein Fibulin-3 promotes KISS1R induced triple negative breast cancer cell invasion.

Breast cancer is a leading cause of cancer mortality. In particular, triple negative breast cancer (TNBC) comprise a heterogeneous group of basal-like tumors lacking estrogen receptor (ERα), progesterone receptor (PR) and HER2 (ErbB2). TNBC represents 15-20% of all breast cancers and occurs frequently in women under 50 years of age. Unfortunately, these patients lack targeted therapy, are typically high grade and metastatic at time of diagnosis. The mechanisms regulating metastasis remain poorly understood. We have previously shown that the kisspeptin receptor, KISS1R stimulates invasiveness of TNBC cells. In this report, we demonstrate that KISS1R signals via the secreted extracellular matrix protein, fibulin-3, to regulate TNBC invasion. We found that the fibulin-3 gene is amplified in TNBC primary tumors and that plasma fibulin-3 levels are elevated in TNBC patients compared to healthy subjects. In this study, we show that KISS1R activation increases fibulin-3 expression and secretion. We show that fibulin-3 regulates TNBC metastasis in a mouse experimental metastasis xenograft model and signals downstream of KISS1R to stimulate TNBC invasion, by activating matrix metalloproteinase 9 (MMP-9) and the MAPK pathway. These results identify fibulin-3 as a new downstream mediator of KISS1R signaling and as a potential biomarker for TNBC progression and metastasis, thus revealing KISS1R and fibulin-3 as novel drug targets in TNBC.

Role of osteopontin as a predictive biomarker for anti-EGFR therapy in triple-negative breast cancer.

OBJECTIVE: Effective targeted therapies for patients with triple-negative breast cancer (TNBC) present an unmet clinical need. There is evidence that TNBCs often have increased expression of the epidermal growth factor receptor (EGFR) and of osteopontin (OPN). OPN-mediated signaling can activate EGFR-dependent signaling pathways. Here, we assessed OPN as a potential predictive biomarker for response to anti-EGFR therapy in TNBC. RESEARCH DESIGN AND METHODS: Using two different TNBC cell lines, MDA-MB-468 and MDA-MB-231, we investigated the impact of stable expression of OPN on efficacy of the EGFR inhibitor erlotinib in vitro. RESULTS: We observed that breast cancer cells engineered to overexpress OPN are more sensitive to growth inhibition by erlotinib than control cells. The level of response was related to the level of OPN expression, possibly due to increased phosphorylation status of EGFR Tyr1068. CONCLUSIONS: These results indicate that OPN expression levels are related to sensitivity of TNBC cells to growth inhibition by erlotinib. OPN thus is a promising predictive biomarker for anti-EGFR therapy in breast cancer.

Genome-wide analysis reveals a role for TDG in estrogen receptor-mediated enhancer RNA transcription and 3-dimensional reorganization.

BACKGROUND: The estrogen receptor (ER) is a ligand-dependant transcription factor expressed in many breast cancers and is the target of many endocrine-based cancer therapies. Genome-wide studies have shown that the ER binds to gene-specific enhancer regions in response to ß-estradiol (E2) which undergo transcription producing noncoding enhancer RNA (eRNA). While eRNAs are important for transcriptional activation of neighboring genes, the mechanism remains poorly understood. RESULTS: Using ChIP-Seq we generate a global profile of thymine DNA glycosylase (TDG), an ER coactivator that plays an essential role in DNA demethylation, in response to E2 in the MCF7 breast cancer cell line. Remarkably, we found that in response to E2 TDG localized to enhancers which also recruit ERα, RNA Pol II and other coregulators and which are marked by histone modifications indicative of active enhancers. Importantly, depletion of TDG inhibits E2-mediated transcription of eRNAs and transcription of ER-target genes. Functionally, we find that TDG both sensitizes MCF7 cells to tamoxifen-mediated cytostasis and increases migration and invasion of MCF7 cells. CONCLUSIONS: Taken together we find that TDG plays a central role in mediating transcription at a subset of enhancers and governs how MCF7 cells respond to both estrogenic and anti-estrogenic compounds and may be an effective therapeutic target.

Concurrent Neoadjuvant Chemotherapy and Radiation Therapy in Locally Advanced Breast Cancer.

PURPOSE: To evaluate whether concurrent neoadjuvant radiation added to standard chemotherapy could increase the pathologic complete response (pCR) to treatment for locally advanced breast cancer (LABC). METHODS AND MATERIALS: This prospective phase 2 trial recruited 32 LABC patients from 2009 to 2011. Patients received neoadjuvant every-3-weekly 5-fluorouracil (500 mg/m2), epirubicin (100 mg/m2), and cyclophosphamide (500 mg/m2) for 3 cycles, followed by weekly docetaxel (35 mg/m2) for 9 cycles. Regional radiation (45 Gy/25 plus 5.4 Gy/5) was delivered concurrently with docetaxel, then modified radical mastectomy. Patients were matched post hoc by a blinded statistician to a concurrent cohort treated with neoadjuvant chemotherapy, modified radical mastectomy, and adjuvant regional radiation. RESULTS: Thirty of 32 patients completed treatment. Twenty-seven were successfully matched by propensity score to 81 control patients by age, stage, and molecular subtype. The concurrent chemoradiation produced a significant increase in pCR (14% vs 22%, P<.001) but no statistically significant difference in disease-free and overall survival at 3 years (respectively, 69% vs 81%, P=.186, hazard ratio 0.51; and 74% vs 89%, P=.162, hazard ratio 0.46). Toxicity included 25% of patients with grade 3 pneumonitis and 25% of patients with dermatitis, and 1 death. CONCLUSIONS: Concurrent neoadjuvant radiation added to radiosensitizing chemotherapy significantly improved pCR. A prospective randomized clinical trial is warranted to exploit the improved response seen with concurrent therapy but using another radio-sensitizing taxane, to better minimize treatment-related toxicity and determine its impact on overall survival.

G protein-coupled KISS1 receptor is overexpressed in triple negative breast cancer and promotes drug resistance.

Triple-negative breast cancer (TNBC) lacks the expression of estrogen receptor α, progesterone receptor and human epidermal growth factor receptor 2 (HER2). TNBC patients lack targeted therapies, as they fail to respond to endocrine and anti-HER2 therapy. Prognosis for this aggressive cancer subtype is poor and survival is limited due to the development of resistance to available chemotherapies and resultant metastases. The mechanisms regulating tumor resistance are poorly understood. Here we demonstrate that the G protein-coupled kisspeptin receptor (KISS1R) promotes drug resistance in TNBC cells. KISS1R binds kisspeptins, peptide products of the KISS1 gene and in numerous cancers, this signaling pathway plays anti-metastatic roles. However, in TNBC, KISS1R promotes tumor invasion. We show that KISS1 and KISS1R mRNA and KISS1R protein are upregulated in TNBC tumors, compared to normal breast tissue. KISS1R signaling promotes drug resistance by increasing the expression of efflux drug transporter, breast cancer resistance protein (BCRP) and by inducing the activity and transcription of the receptor tyrosine kinase, AXL. BCRP and AXL transcripts are elevated in TNBC tumors, compared to normal breast, and TNBC tumors expressing KISS1R also express AXL and BCRP. Thus, KISS1R represents a potentially novel therapeutic target to restore drug sensitivity in TNBC patients.

The transcriptional regulator TBX3 promotes progression from non-invasive to invasive breast cancer.

BACKGROUND: TBX3 is a T-box transcription factor repressor that is elevated in metastatic breast cancer and is believed to promote malignancy of tumor cells, possibly by promoting cell survival and epithelial-mesenchymal transition. METHODS: The relative expression of TBX3 was assessed in the 21T cell lines which were derived from an individual patient and represent three distinct stages of breast cancer progression: 21PT, atypical ductal hyperplasia; 21NT, ductal carcinoma in situ; and 21MT-1, invasive mammary carcinoma. Two different isoforms of TBX3 (TBX3iso1 and TBX3iso2) were overexpressed to evaluate cell survival/colony forming ability, growth, and invasion in the ductal carcinoma in situ-like 21NT cell line using an in vitro Matrigel model of cancer progression. In addition, TBX3 expression was knocked down to evaluate the effects of downregulating TBX3 on the invasive mammary carcinoma-like 21MT-1 cell line. Finally, PCR array profiling was used to assess alterations in gene expression due to TBX3 overexpression in the 21NT cells. RESULTS: TBX3 is abundant in the invasive 21MT-1 cell line, while being minimally expressed in the non-invasive 21NT and 21PT cell lines. Overexpression of either TBX3iso1 or TBX3iso2 in 21NT cells resulted in increased cell survival/colony forming ability, growth vs. apoptosis and invasion in Matrigel. In contrast, short hairpin RNA-mediated knockdown of TBX3 in the 21MT-1 cells resulted in smaller colonies, with a more regular, less dispersed (less infiltrative) morphology. Array profiling of the 21NT TBX3 iso1 and iso2 transfectants showed that there are common alterations in expression of several genes involved in signal transduction, cell cycle control/cell survival, epithelial-mesenchymal transition and invasiveness. CONCLUSIONS: Overall, these results indicate that TBX3 (isoform 1 or 2) expression can promote progression in a model of early breast cancer by altering cell properties involved in cell survival/colony formation and invasiveness, as well as key regulatory and EMT/invasiveness-related gene expressions.

Primary melanoma tumor inhibits metastasis through alterations in systemic hemostasis.

Progression from a primary tumor to distant metastases requires extensive interactions between tumor cells and their microenvironment. The primary tumor is not only the source of metastatic cells but also can also modulate host responses to these cells, leading to an enhancement or inhibition of metastasis. Tumor-mediated stimulation of bone marrow can result in pre-metastatic niche formation and increased metastasis. However, a primary tumor can also inhibit metastasis through concomitant tumor resistance-inhibition of metastatic growth by existing tumor mass. Here, we report that the presence of a B16F10 primary tumor significantly restricted numbers and sizes of experimental lung metastases through reduction of circulating platelets and reduced formation of metastatic tumor cell-associated thrombi. Tumor-bearing mice displayed splenomegaly, correlated with primary tumor size and platelet count. Reduction in platelet numbers in tumor-bearing animals was responsible for metastatic inhibition, as restoration of platelet numbers using isolated platelets re-established both tumor cell-associated thrombus formation and experimental metastasis. Consumption of platelets due to a B16F10 primary tumor is a form of concomitant tumor resistance and demonstrates the systemic impact of a growing tumor. Understanding the interplay between primary tumors and metastases is essential, as clarification of concomitant tumor resistance mechanisms may allow inhibition of metastatic growth following tumor resection. Key messages Mice with a primary B16F10 tumor had reduced metastasis vs. mice without a primary tumor. Tumor-bearing mice had splenomegaly and fewer platelets and tumor-associated thrombi. Restoring platelets restored tumor-associated thrombi and increased metastasis. This work shows the impact that a primary tumor can have on systemic metastasis. Understanding these interactions may lead to improved ways to inhibit metastasis.

Role of plasma osteopontin as a biomarker in locally advanced breast cancer.

Osteopontin (OPN), a malignancy-associated secreted phosphoprotein, is a prognostic plasma biomarker for survival in metastatic breast cancer patients. We evaluated the role of OPN in Locally Advanced Breast Cancer (LABC) patients in predicting response to neoadjuvant chemotherapy and association with survival. Fifty-three patients with non-metastatic LABC were enrolled in this study and monitored serially for plasma OPN levels by ELISA during neoadjuvant chemotherapy prior to surgery. For fifty patients who had baseline OPN levels available for analysis, the median baseline OPN level was 63.6 ng/ml. Median patient follow up was 45 months and thirteen patients died from metastatic disease. Patients with baseline OPN levels ≥ 63.6 ng/ml were significantly more likely to die of their disease than those with baseline OPN < 63.6 ng/mL (Hazard Ratio = 3.4; 95% confidence interval 1.4-11.3; P = 0.011), and overall, baseline OPN level was significantly associated with survival (P = 0.002). There was little support for value of serial OPN determination in monitoring response to therapy in this patient population. Although the percentage of patients with baseline OPN levels < 63.6 ng/ml was higher in patients with pathological complete response than in those with no response, the difference was not statistically significant (64% and 14%, respectively (P = 0.066)). Thus, baseline plasma OPN level is a prognostic biomarker in this group of LABC patients, and could also be helpful in identifying LABC patients who will respond to neoadjuvant chemotherapy. Our results call for validation of our findings in large prospective trial data sets.

Stage of breast cancer progression influences cellular response to activation of the WNT/planar cell polarity pathway.

Planar cell polarity (PCP) signaling has been shown in different studies to either promote or inhibit the malignancy of breast cancer. Using the 21T cell lines, which were derived from an individual patient and represent distinct stages of progression, we show that the prototypical PCP ligand, WNT5A, is expressed highest in 21MT-1 cells (invasive mammary carcinoma) and lowest in 21PT (atypical ductal hyperplasia) and 21NT (ductal carcinoma in situ) cells. Overexpression of WNT5A decreased spherical colony formation and increased invasion and in vivo extravasation only in 21NT cells; whereas overexpression increased migration of both 21PT and 21NT cells. WNT5A overexpression also increased RHOA expression of both cell lines and subsequent RHOA knockdown blocked WNT5A-induced migration, but only partially blocked WNT5A-induced invasion of 21NT cells. PCP can signal through VANGL1 to modulate AP-1 target genes (e.g. MMP3) and induce invasion. VANGL1 knockdown inhibited WNT5A-induced invasion of 21NT cells, but had no effect on WNT5A-induced migration of either 21PT or 21NT cells. WNT5A-induced MMP3 expression was seen only in 21NT cells, an effect that was VANGL1 dependent, but independent of AP-1. We thus provide evidence that PCP signaling can act in a context dependent manner to promote breast cancer progression.

Assessment of osteopontin in early breast cancer: correlative study in a randomised clinical trial.

INTRODUCTION: Osteopontin (OPN) is a malignancy-associated glycoprotein that contributes functionally to tumor aggressiveness. In metastatic breast cancer, we previously demonstrated that elevated OPN in primary tumor and blood was associated with poor prognosis. METHODS: We measured OPN in plasma by ELISA, and in tumors by immunohistochemistry, in 624 (94%) and 462 (69%), respectively, of 667 postmenopausal women with hormone responsive early breast cancer treated by surgery followed by adjuvant treatment with tamoxifen +/- octreotide in a randomized trial (NCIC CTG MA.14; National Cancer Institute of Canada Clinical Trials Group Mammary.14). RESULTS: Plasma OPN was measured in 2,540 samples; 688 at baseline and 1,852 collected during follow-up. Mean baseline plasma OPN was 46 ng/ml (range 22.6 to 290) which did not differ from normal levels. Mean percentage OPN tumor cell positivity was 33.9 (95% CI: 30.2 to 37.9). There was no correlation between plasma and tumor OPN values. In multivariate analysis, neither was associated with event-free survival (EFS), relapse-free survival (RFS), overall survival (OS), bone RFS or non-bone RFS. An exploratory analysis in patients with recurrence showed higher mean OPN plasma levels 60.7 ng/ml (23.9 to 543) in the recurrence period compared with baseline levels. CONCLUSIONS: The hypothesis that OPN tumor expression would have independent prognostic value in early breast cancer was not supported by multivariate analysis of this study population. Plasma OPN levels in women with hormone responsive early breast cancer in the MA.14 trial were not elevated and there was no evidence for prognostic value of plasma OPN in this defined group of patients. However, our finding of elevated mean OPN plasma level around the time of recurrence warrants further study. TRIAL REGISTRATION: NCT00002864, http://clinicaltrials.gov/show/NCT00002864.

Osteopontin increases breast cancer cell sensitivity to specific signaling pathway inhibitors in preclinical models.

Src kinase and Hsp90 play important roles in malignancy-promoting signaling pathways in a variety of cancers and related targeting agents are presently in clinical trials. To help improve the success of Src kinase and Hsp90 inhibitor therapies, identifying sensitive patient populations will be essential. Osteopontin (OPN), a secreted integrin-binding glycophosphoprotein, is associated with progression and metastasis in a variety of cancers and has been studied as a prognostic marker. Previous work of ours and other groups has indicated that OPN-induced signal transduction involves activation of both Src and Hsp90-dependent pathways. We thus hypothesized that over-expression of OPN could make tumor cells more vulnerable to these classes of inhibitors. This study used multiple in vitro assays to determine if OPN levels could predict breast cancer cell sensitivity to Hsp90 and/or Src kinase inhibitors. We used multiple derivatives of two unrelated of human breast cancer cell lines, high vs. low levels of OPN, to determine if OPN affects the response to two specific inhibitors, an Hsp90 inhibitor and a Src kinase inhibitor, in in vitro migration and colony formation assays. Cells had greater decreases in migration and colony forming ability after Hsp90 and Src kinase inhibitor treatments when OPN was present (either endogenous or exogenous). Decreasing OPN levels via shRNA knockdown decreased inhibitor effects. In rescue experiments, adding exogenous OPN to non-expressing cells increased inhibitor effects. These results suggest that OPN could potentially be useful clinically as a predictive marker in identifying patients who will benefit from either Hsp90 or Src kinase inhibitor therapy.

Nuclear localization of maspin is essential for its inhibition of tumor growth and metastasis.

Maspin (mammary serine protease inhibitor or SerpinB5) acts as a tumor suppressor when overexpressed in aggressive cancer cell lines. However, its role in human cancer is controversial. Maspin expression has been associated with a poor prognosis in some studies, whereas in others, with favorable outcome. The clinical data suggest, however, that nuclear-localized maspin is associated with improved survival. We hypothesized that the tumor suppressor activity of maspin may require nuclear localization, and that the discordance between clinical and experimental reports is a consequence of the variable subcellular distribution of maspin. Furthermore, we surmized that nuclear maspin could function as a tumor suppressor through the regulation of genes involved in tumor growth and invasion. Maspin or maspin fused to a nuclear export signal were expressed in metastatic human breast and epidermoid carcinoma cell lines. We found that pan-cellular localized maspin inhibited in vivo tumor growth and metastasis when assessed in xenograft chicken embryo and murine mammary fat pad injection models. However, when maspin was excluded from the nucleus via a nuclear exclusion signal, it no longer functioned as a metastasis suppressor. Using chromatin immunoprecipitation, we show that nuclear maspin was enriched at the promoter of colony-stimulating factor-1 (CSF-1) and associated with diminished levels of CSF-1 mRNA. Our findings demonstrate that the nuclear localization of maspin is required for its tumor and metastasis suppressor functions in vivo, and suggest that its mechanism of action involves, in part, direct association of maspin with target genes.

Pre- and post-translational regulation of osteopontin in cancer.

Osteopontin (OPN) is a matricellular protein that binds to a number of cell surface receptors including integrins and CD44. It is expressed in many tissues and secreted into body fluids including blood, milk and urine. OPN plays important physiological roles in bone remodeling, immune response and inflammation. It is also a tumour-associated protein, and elevated OPN levels are associated with tumour formation, progression and metastasis. Research has revealed a promising role for OPN as a cancer biomarker. OPN is subject to alternative splicing, as well as post-translational modifications such as phosphorylation, glycosylation and proteolytic cleavage. Functional differences have been revealed for different isoforms and post-translational modifications. The pattern of isoform expression and post-translational modification is cell-type specific and may influence the potential role of OPN in malignancy and as a cancer biomarker.

Role of the metastasis-promoting protein osteopontin in the tumour microenvironment.

Osteopontin (OPN) is a secreted protein present in bodily fluids and tissues. It is subject to multiple post-translational modifications, including phosphorylation, glycosylation, proteolytic cleavage and crosslinking by transglutamination. Binding of OPN to integrin and CD44 receptors regulates signalling cascades that affect processes such as adhesion, migration, invasion, chemotaxis and cell survival. A variety of cells and tissues express OPN, including bone, vasculature, kidney, inflammatory cells and numerous secretory epithelia. Normal physiological roles include regulation of immune functions, vascular remodelling, wound repair and developmental processes. OPN also is expressed in many cancers, and elevated levels in patients' tumour tissue and blood are associated with poor prognosis. Tumour growth is regulated by interactions between tumour cells and their tissue microenvironment. Within a tumour mass, OPN can be expressed by both tumour cells and cellular components of the tumour microenvironment, and both tumour and normal cells may have receptors able to bind to OPN. OPN can also be found as a component of the extracellular matrix. The functional roles of OPN in a tumour are thus complex, with OPN secreted by both tumour cells and cells in the tumour microenvironment, both of which can in turn respond to OPN. Much remains to be learned about the cross-talk between normal and tumour cells within a tumour, and the role of multiple forms of OPN in these interactions. Understanding OPN-mediated interactions within a tumour will be important for the development of therapeutic strategies to target OPN.

Human 21T breast epithelial cell lines mimic breast cancer progression in vivo and in vitro and show stage-specific gene expression patterns.

Early breast cancer progression involves advancement through specific morphological stages including atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (IMC), although not necessarily always in a linear fashion. Observational studies have examined genetic, epigenetic and gene expression differences in breast tissues representing these stages of progression, but model systems which would allow for experimental testing of specific factors influencing transition through these stages are scarce. The 21T series cell lines, all originally derived from the same patient with metastatic breast cancer, have been proposed to represent a mammary tumor progression series. We report here that three of the 21T cell lines indeed mimic specific stages of human breast cancer progression (21PT-derived cells, ADH; 21NT-derived cells, DCIS; 21MT-1 cells, IMC) when grown in the mammary fat pad of nude mice, albeit after a year. To develop a more rapid, readily manipulatable in vitro assay for examining the biological differences between these cell lines, we have used a 3D Matrigel system. When the three cell lines were grown in 3D Matrigel, they showed characteristic morphologies, in which quantifiable aspects of stage-specific in vivo behaviors (ie, differences in acinar structure formation, cell polarization, colony morphology, cell proliferation, cell invasion) were recapitulated in a reproducible fashion. Gene expression profiling revealed a characteristic pattern for each of the three cell lines. Interestingly, Wnt pathway alterations are particularly predominant in the early transition from 21PTci (ADH) to 21NTci (DCIS), whereas alterations in expression of genes associated with control of cell motility and invasion phenomena are more prominent in the later transition of 21NTci (DCIS) to 21MT-1 (IMC). This system thus reveals potential therapeutic targets and will provide a means of testing the influences of identified genes on transitions between these stages of pre-malignant to malignant growth.