Remodeling of purinergic receptor-mediated Ca 2+ signaling as a consequence of EGF-induced epithelial-mesenchymal transition in breast cancer cells

Background The microenvironment plays a pivotal role in tumor cell proliferation, survival and migration. Invasive cancer cells face a new set of environmental challenges as they breach the basement membrane and colonize distant organs during the process of metastasis. Phenotypic switching, such as that which occurs during epithelial-mesenchymal transition (EMT), may be associated with a remodeling of cell surface receptors and thus altered responses to signals from the tumor microenvironment. Methodology/Principal Findings We assessed changes in intracellular Ca 2+ in cells loaded with Fluo-4 AM using a fluorometric imaging plate reader (FLIPR TETRA) and observed significant changes in the potency of ATP (EC 50 0.175 μM (-EGF) versus 1.731 μM (+EGF), P<0.05), and the nature of the ATP-induced Ca 2+ transient, corresponding with a 10-fold increase in the mesenchymal marker vimentin (P<0.05). We observed no change in the sensitivity to PAR2-mediated Ca 2+ signaling, indicating that these alterations are not simply a consequence of changes in global Ca 2+ homeostasis. To determine whether changes in ATP-mediated Ca 2+ signaling are preceded by alterations in the transcriptional profile of purinergic receptors, we analyzed the expression of a panel of P2X ionotropic and P2Y metabotropic purinergic receptors using real-time RT-PCR and found significant and specific alterations in the suite of ATP-activated purinergic receptors during EGF-induced EMT in breast cancer cells. Our studies are the first to show that P2X 5 ionotropic receptors are enriched in the mesenchymal phenotype and that silencing of P2X 5 leads to a significant reduction (25%, P<0.05) in EGF-induced vimentin protein expression. Conclusions The acquisition of a new suite of cell surface purinergic receptors is a feature of EGF-mediated EMT in MDA-MB-468 breast cancer cells. Such changes may impart advantageous phenotypic traits and represent a novel mechanism for the targeting of cancer metastasis.

The relative risk of second primary cancers in Queensland, Australia: A retrospective cohort study

Background Cancer survivors face an increased likelihood of being subsequently diagnosed with another cancer. The aim of this study was to quantify the relative risk of survivors developing a second primary cancer in Queensland, Australia. Methods Standardised incidence rates stratified by type of first primary cancer, type of second primary cancer, sex, age at first diagnosis, period of first diagnosis and follow-up interval were calculated for residents of Queensland, Australia, who were diagnosed with a first primary invasive cancer between 1982 and 2001 and survived for a minimum of 2 months. Results A total of 23,580 second invasive primary cancers were observed over 1,370,247 years of follow-up among 204,962 cancer patients. Both males (SIR = 1.22; 95% CI = 1.20-1.24) and females (SIR = 1.36; 95% CI = 1.33-1.39) within the study cohort were found to have a significant excess risk of developing a second cancer relative to the incidence of cancer in the general population. The observed number of second primary cancers was also higher than expected within each age group, across all time periods and during each follow-up interval. Conclusions The excess risk of developing a second malignancy among cancer survivors can likely be attributed to factors including similar aetiologies, genetics and the effects of treatment, underlining the need for ongoing monitoring of cancer patients to detect subsequent tumours at an early stage. Education campaigns developed specifically for survivors may be required to lessen the prevalence of known cancer risk factors.

Therapeutic immunisation of rabbits with cottontail rabbit papillomavirus (CRPV) virus-like particles (VLP) induces regression of established papillomas

There is overwhelming evidence that persistent infection with high-risk human papillomaviruses (HR-HPV) is the main risk factor for invasive cancer of the cervix. Due to this global public health burden, two prophylactic HPV L1 virus-like particles (VLP) vaccines have been developed. While these vaccines have demonstrated excellent type-specific prevention of infection by the homologous vaccine types (high and low risk HPV types), no data have been reported on the therapeutic effects in people already infected with the low-risk HPV type. In this study we explored whether regression of CRPV-induced papillomas could be achieved following immunisation of out-bred New Zealand White rabbits with CRPV VLPs. Rabbits immunised with CRPV VLPs had papillomas that were significantly smaller compared to the negative control rabbit group (P ≤ 0.05). This data demonstrates the therapeutic potential of PV VLPs in a well-understood animal model with potential important implications for human therapeutic vaccination for low-risk HPVs. © 2008 Govan et al; licensee BioMed Central Ltd.

Circulating tumour cells in metastatic head and neck cancers

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer with 650,000 new cases p/a worldwide. HNSCC causes high morbidity with a 5-year survival rate of less than 60%, which has not improved due to the lack of early detection (Bozec et al. Eur Arch Otorhinolaryngol. 2013;270: 2745–9). Metastatic disease remains one of the leading causes of death in HNSCC patients. This review article provides a comprehensive overview of literature over the past 5 years on the detection of circulating tumour cells (CTCs) in HNSCC; CTC biology and future perspectives. CTCs are a hallmark of invasive cancer cells and key to metastasis. CTCs can be used as surrogate markers of overall survival and progression-free survival. CTCs are currently used as prognostic factors for breast, prostate and colorectal cancers using the CellSearch® system. CTCs have been detected in HNSCC, however, these numbers depend on the technique applied, time of blood collection and the clinical stage of the patient. The impact of CTCs in HNSCC is not well understood, and thus, not in routine clinical practice. Validated detection technologies that are able to capture CTCs undergoing epithelial–mesenchymal transition are needed. This will aid in the capture of heterogeneous CTCs, which can be compiled as new targets for the current food and drug administration-cleared CellSearch® system. Recent studies on CTCs in HNSCC with the CellSearch® have shown variable data. Therefore, there is an immediate need for large clinical trials encompassing a suite of biomarkers capturing CTCs in HNSCC, before CTCs can be used as prognostic markers in HNSCC patient management.

Depression, anxiety and body image after treatment for invasive stage one epithelial ovarian cancer

Background: Diagnosis of epithelial ovarian cancer (EOC) in young women has major implications including those to their reproductive potential. We evaluated depression, anxiety and body image in patients with stage I EOC treated with fertility sparing surgery (FSS) or radical surgery (RS). We also investigated fertility outcomes after FSS.----- Methods: A retrospective study was undertaken in which 62 patients completed questionnaires related to anxiety, depression, body image and fertility outcomes. Additional information on adjuvant therapy after FSS and RS and demographic details were abstracted from medical records. Both bi and multivariate regression models were used to assess the relationship between demographic, clinical and pathological results and scores for anxiety, depression and body image.----- Results: Thirty-nine patients underwent RS and the rest, FSS. The percentage of patients reporting elevated anxiety and depression (subscores ≥ 11) were 27 % and 5% respectively. The median (inter quartile range) score for body image scale (BIS) was 6 (3-15). None of the demographic or clinical factors examined showed significant association with anxiety and BIS with the exception of ‘time since diagnosis’. For depression, post-menopausal status was the only independent predictor. Among those 23 patients treated by FSS, 14 patients tried to conceive (7 successful), resulting in 7 live births, one termination of pregnancy and one miscarriage.----- Conclusion: This study shows that psychological issues are common in women treated for stage I EOC. Reproduction after FSS is feasible and lead to the birth of healthy babies in about half of patients who wished to have another child. Further prospective studies with standardised instruments are required.

Carbonic anhydrase IX expression and outcome after radiotherapy for muscle-invasive bladder cancer

Aims: Carbonic anhydrase IX (CA IX) expression has been described as an endogenous marker of hypoxia in solid neoplasms. Furthermore, CA IX expression has been associated with an aggressive phenotype and resistance to radiotherapy. We assessed the prognostic significance of CA IX expression in patients with muscle-invasive bladder cancer treated with radiotherapy. Materials and methods: A standard immunohistochemistry technique was used to show CA IX expression in 110 muscle-invasive bladder tumours treated with radiotherapy. Clinicopathological data were obtained from medical case notes. Results: CA IX immunostaining was detected in 89 (∼81%) patients. Staining was predominantly membranous, with areas of concurrent cytoplasmic and nuclear staining and was abundant in luminal and perinecrotic areas. No significant correlation was shown between the overall CA IX status and the initial response to radiotherapy, 5-year bladder cancer-specific survival or the time to local recurrence. Conclusions: The distribution of CA IX expression in paraffin-embedded tissue sections seen in this series is consistent with previous studies in bladder cancer, but does not provide significant prognostic information with respect to the response to radiotherapy at 3 months and disease-specific survival after radical radiotherapy. © 2007 The Royal College of Radiologists.

The invasive and metastatic properties of hormone-independent but hormone-responsive variants of MCF-7 human breast cancer cells

We have previously isolated a series of MCF-7 human breast cancer cell variants which no longer require estrogen-supplementation for tumor growth in nude mice (Clarke et al. Proc Natl Acad Sci USA 86: 3649-3653, 1989). We now report that these hormone-independent and hormone-responsive variants (MIII, MCF7/LCC1) can invade locally from solid mammary fat pad tumors, and produce primary extensions on the surface of intraperitoneal structures including liver, pancreas, and diaphragm. Both lymphatic and hematogenous dissemination are observed, resulting in the establishing of pulmonary, bone, and renal metastases. The pattern of metastasis by MIII and MCF7/LCC1 cells closely resembles that frequently observed in breast cancer patients, and provides the first evidence of metastasis from MCF-7 cells growing in vivo without supplementary estrogen. The interexperimental incidence of metastases, and the time from cell inoculation to the appearance of metastatic disease are variable. The increased metastatic potential is not associated with an increase in either the level of laminin attachment, laminin receptor mRNA expression, or secreted type IV collagenolytic activity. We also did not detect a significant decrease in the steady-state mRNA levels of the metastasis inhibitor nm23 gene. However, when growing without estrogen in vitro, MCF7/LCC1 cells produce elevated levels of the estrogen-inducible cathepsin D enzyme.

The LCC15-MB human breast cancer cell line expresses osteopontin and exhibits an invasive and metastatic phenotype

We have characterized the LCC15-MB cell line which was recently derived from a breast carcinoma metastasis resected from the femur of a 29-year-old woman. LCC15-MB cells are vimentin (VIM) positive, exhibit a stellate morphology in routine cell culture, and form penetrating colonies when embedded in three-dimensional gels of Matrigel or fibrillar collagen. They show high levels of activity in the Boyden chamber chemomigration and chemoinvasion assays, and like other invasive human breast cancer (HBC) cell lines, LCC15-MB cells activate matrix-metalloproteinase-2 in response to treatment with concanavalin A. In addition, these cells are tumorigenic when implanted subcutaneously in nude mice and recolonize bone after arterial injection. Interestingly, both the primary lesion and the bone metastasis from which LCC15-MB were derived, as well as the resultant cell line, abundantly express the bone matrix protein osteopontin (OPN). OPN is also expressed by the highly metastatic MDA-MB-435 cells, but not other invasive or noninvasive HBC cell lines. Expression of OPN is retained in the subcutaneous xenograft and intraosseous metastases of LCC15-MB as detected by immunohistochemistry. Both VIM and OPN expression have been associated with breast cancer invasion and metastasis, and their expression by the LCC15-MB cell line is consistent with its derivation from a highly aggressive breast cancer. These cells provide a useful model for studying molecular mechanisms important for breast cancer metastasis to bone and, in particular, the implication(s) of OPN and VIM expression in this process.

Cell adhesion molecule uvomorulin expression in human breast cancer cell lines : relationship to morphology and invasive capacities

Loss of cell-cell adhesion in carcinoma cells may be an important step in the acquisition of an invasive, metastatic phenotype. We have examined the expression of the epithelial-specific cell adhesion molecule uvomorulin (E-cadherin, cell-CAM 120/80, L-CAM) in human breast cancer cell lines. We find that fibroblastoid, highly invasive, vimentin-expressing breast cancer cell lines do not express uvomorulin. Of the more epithelial-appearing, less invasive, keratin-expressing breast cancer cell lines, some express uvomorulin, and some do not. We examined the morphologies of the cell lines in the reconstituted basement membrane matrix Matrigel and measured the ability of the cells to traverse a Matrigel-coated filter as in vitro models for detachment of carcinoma cells from neighboring cells and invasion through basement membrane into surrounding tissue. Colonies of uvomorulin-positive cells have a characteristic fused appearance in Matrigel, whereas uvomorulin-negative cells appear detached. Cells which are uvomorulin negative and vimentin positive have a stellate morphology in Matrigel. We show that uvomorulin is responsible for the fused colony morphology in Matrigel since treatment of uvomorulin-positive MCF-7 cells with an antibody to uvomorulin caused the cells to detach from one another but did not induce invasiveness in these cells, as measured by their ability to cross a Matrigel-coated polycarbonate filter in a modified Boyden chamber assay. Two uvomorulin-negative, vimentin-negative cell lines are also not highly invasive as measured by this assay. We suggest that loss of uvomorulin-mediated cell-cell adhesion may be one of many changes involved in the progression of a carcinoma cell to an invasive phenotype.

Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44 : correlation with invasive potential

In the present study, we examined a panel of human breast cancer cell lines with regard to their expression of CD44 and ability to bind and degrade hyaluronan. The cell lines expressed varying amounts of different molecular weight forms of CD44 (85-200 kDa) and, in general, those that expressed the greatest amounts of CD44 were the most invasive as judged by in vitro assays. In addition, the ability to bind and degrade hyaluronan was restricted to the cell lines expressing high levels of CD44, and both these functions were blocked by an antibody to CD44 (Hermes-1). Moreover, the rate of [3H]hyaluronan degradation was highly correlated with the amount of CD44 (r = 0.951, P < 0.0001), as well as with the invasive potential of the cells. Scatchard analysis of the [3H]hyaluronan binding of these cells revealed the existence of significant differences in both their binding capacity and their dissociation constant. To determine the source of this deviation, the different molecular weight forms of CD44 were partially separated by gel filtration chromatography. In all cell lines, the 85 kDa form was able to bind hyaluronan, although with different affinities. In contrast, not all of the high molecular weight forms of CD44 had this ability. These results illustrate the diversity of CD44 molecules in invasive tumor cells, and suggest that one of their major functions is to degrade hyaluronan.

Acquisition of estrogen independence and antiestrogen resistance in breast cancer : association with the invasive and metastatic phenotype

A significant percentage of human breast cancer (HBC) is dependent upon the ovarian hormone estrogen for its onset and progression. The presence or lack of estrogen receptors (ERs) in human breast cancer is an important determinant both of prognosis and of choice of treatment - a poorer prognosis being associated with ER–ve disease. Cell lines established from human breast cancer provide models for breast cancer in various stages of progression (Engel & Young 1978). When grown as tumors in athymic nude mice, these lines represent the major in vivo experimental model for HBC studies (Brünner et al 1987). The ease of both in vitro and in vivo maintenance, the human derivation of the tissue, and the similarities in plasma estrogen levels between ovariectomized nude mice and postmenopausal women (Seibert et al. 1983, Brünner et al. 1986), make the growth of human breast cancer cell lines in nude mice an attractive...

Heregulin regulates the actin cytoskeleton and promotes invasive properties in breast cancer cell lines

The metastatic process requires changes in tumor cell adhesion properties, cell motility and remodeling of the extracellular matrix. The erbB2 proto-oncogene is overexpressed in approximately 30% of breast cancers and is a major prognostic parameter when present in invasive disease. A ligand for the erbB2 receptor has not yet been identified but it can be activated by heterodimerization with heregulin (HRG)-stimulated erbB3 and erbB4 receptors. The HRGs are a family of polypeptide growth factors that have been shown to play a role in embryogenesis, tumor formation, growth and differentiation of breast cancer cells. The erbB3 and erbB4 receptors are involved in transregulation of erbB2 signaling. The work presented here suggests biological roles for HRG including regulation of the actin cytoskeleton and induction of motility and invasion in breast cancer cells. HRG-expressing breast cancer cell lines are characterized by low erbB receptor levels and a high invasive and metastatic index, while those which overexpress erbB2 demonstrate minimal invasive potential in vitro and are non-tumorigenic in vivo. Treatment of the highly tumorigenic and metastatic HRG-expressing breast cancer cell line MDA-MB-231 with an HRG-neutralizing antibody significantly inhibited proliferation in culture and motility in the Boyden chamber assay. Addition of exogenous HRG to non-invasive erbB2 overexpressing cells (SKBr-3) at low concentrations induced formation of pseudopodia, enhanced phagocytic activity and increased chemomigration and invasion in the Boyden chamber assay. The specificity of the chemomigration response to HRG is demonstrated by inhibition with the anti-HRG neutralizing antibody. These results suggest that either HRG can act as an autocrine or paracrine ligand to promote the invasive behavior of breast cancer cells in vitro or thus may enhance the metastatic process in vivo.

Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models

Approximately half of prostate cancers (PCa) carry TMPRSS2-ERG translocations; however, the clinical impact of this genomic alteration remains enigmatic. Expression of v-ets erythroblastosis virus E26 oncogene like (avian) gene (ERG) promotes prostatic epithelial dysplasia in transgenic mice and acquisition of epithelial-to-mesenchymal transition (EMT) characteristics in human prostatic epithelial cells (PrECs). To explore whether ERG-induced EMT in PrECs was associated with therapeutically targetable transformation characteristics, we established stable populations of BPH-1, PNT1B and RWPE-1 immortalized human PrEC lines that constitutively express flag-tagged ERG3 (fERG). All fERG-expressing populations exhibited characteristics of in vitro and in vivo transformation. Microarray analysis revealed >2000 commonly dysregulated genes in the fERG-PrEC lines. Functional analysis revealed evidence that fERG cells underwent EMT and acquired invasive characteristics. The fERG-induced EMT transcript signature was exemplified by suppressed expression of E-cadherin and keratins 5, 8, 14 and 18; elevated expression of N-cadherin, N-cadherin 2 and vimentin, and of the EMT transcriptional regulators Snail, Zeb1 and Zeb2, and lymphoid enhancer-binding factor-1 (LEF-1). In BPH-1 and RWPE-1-fERG cells, fERG expression is correlated with increased expression of integrin-linked kinase (ILK) and its downstream effectors Snail and LEF-1. Interfering RNA suppression of ERG decreased expression of ILK, Snail and LEF-1, whereas small interfering RNA suppression of ILK did not alter fERG expression. Interfering RNA suppression of ERG or ILK impaired fERG-PrEC Matrigel invasion. Treating fERG-BPH-1 cells with the small molecule ILK inhibitor, QLT-0267, resulted in dose-dependent suppression of Snail and LEF-1 expression, Matrigel invasion and reversion of anchorage-independent growth. These results suggest that ILK is a therapeutically targetable mediator of ERG-induced EMT and transformation in PCa.