Is there a malignant progression associated with a linear change in protein expression levels from normal canine mammary gland to metastatic mammary tumors?

The molecular mechanisms of the development of canine mammary tumors are still incompletely understood. In the present study we hypothesized that there is a malignant progression from normal gland to malignant carcinomas that is associated with a linear change in protein expression. To this end, the proteome of canine normal mammary gland, adenomas, nonmetastatic carcinomas, and metastatic carcinomas was compared. Application of 2D-DIGE and MALDI-TOF-MS identified 48 proteins with significant changes (fold change >|1.5|; p < 0.05) in expression levels at the different stages of malignant progression. Forty-two of these followed three major stepwise but not linear expression patterns. Thirteen proteins showed the adenoma pattern characterized by a change in protein expression levels during progression from normal gland to adenomas which persisted on the same level at the subsequent stages of malignancy. Nine proteins followed the carcinoma pattern with an up- or down-regulation between adenomas and carcinomas. The majority of 20 proteins followed the metastasis pattern with a significant change of protein expression levels between nonmetastatic and metastatic carcinomas. The present study therefore shows that differences in malignancy are associated with a stepwise but not linear change in protein expression levels, which does not finally confirm or disapprove the existence of a malignant progression in canine mammary tumors. In addition, the acquisition of metastatic potential seems to be associated with the strongest changes in protein expression levels.     

Quantitative proteomics of primary tumors with varying metastatic capabilities using stable isotope-labeled proteins of multiple histogenic origins

The development of metastasis is a complex, multistep process that remains poorly defined. To identify proteins involved in the colonization phase of the metastatic process, we compared the proteome of tumors derived from inoculation of a panel of isogenic human cancer cell lines with different metastatic capabilities into the mammary fat pad of immunodeficient mice. Using a protein standard generated by SILAC-labeling, a total of 675 proteins were identified and 30 were differentially expressed between at least two of the tumors. The protein standard contained the proteomes of seven cell lines from multiple histogenic origins and displayed superior features compared to standard super-SILAC. The expression of some proteins correlated with metastatic capabilities, such as myosin-9 (nonmuscle myosin II A) and L-lactate dehydrogenase A, while the expression of elongation factor tu correlated inversely to metastatic capabilities. The expression of these proteins was biochemically validated, and expression of myosin-9 in clinical breast cancer samples was further shown to be altered in primary tumors versus corresponding lymph node metastasis. Our study demonstrates an improved strategy for quantitative comparison of an unlimited number of tumor tissues, and provides novel insights into key proteins associated with the colonization phase of metastasis formation.     

Identification of breast cancer metastasis-associated proteins in an isogenic tumor metastasis model using two-dimensional gel electrophoresis and liquid chromatography-ion trap-mass spectrometry

To better understand the molecular mechanisms underlying breast cancer metastasis and search for potential markers for metastatic progression, we have developed a highly metastatic variant of human MDA-MB-435 breast cancer cell line through in vivo stepwise selection of pulmonary metastatic cells caused by parental MDA-MB-435 cells in the athymic mice. Comparative proteomic analysis using 2-DE and LC-IT-MS revealed that 102 protein spots were reproducibly altered more than three-fold between the selected variant and its parental counterpart. Eleven differentially expressed protein spots were identified with high confidence using SEQUEST with uninterpreted tandem mass raw data. Cathepsin D precursor, peroxiredoxin 6 (PDX6), heat shock protein 27 (HSP27), HSP60, tropomyosin 1 (TPM1), TPM2, TPM3, TPM4, 14-3-3 protein epsilon, and tumor protein D54 were up-regulated in the highly metastatic variant, whereas alpha B-crystalline (CRAB) was only detected in its parental counterpart. Differential expression was confirmed for four proteins including PDX6, CRAB, TPM4, and HSP60 by real-time quantitative PCR and Western blotting analysis in our model. Immunohistochemical analysis in 80 breast cancer donors demonstrated a significant association of TPM4 (p = 0.002), HSP60 (p = 0.001), PDX6 (p = 0.002) but not CRAB (p = 0.113) staining with the presence of lymph node metastasis. In addition, TPM4 staining was also associated with clinical stage (p = 0.000), but no significant association was found between TPM4, PDX6, CRAB, and HSP60 expression and tumor size, hormone receptor, and HER-2 status (p > 0.05). The functional implication of these identified proteins was also discussed. These proteomic data are valuable and informative for understanding breast cancer metastasis and searching for potential markers for metastatic progression.     

Common plasma protein marker LCAT in aggressive human breast cancer and canine mammary tumor

Breast cancer is one of the most frequently diagnosed cancers. Although biomarkers are continuously being discovered, few specific markers, rather than classification markers, representing the aggressiveness and invasiveness of breast cancer are known. In this study, we used samples from canine mammary tumors in a comparative approach. We subjected 36 fractions of both canine normal and mammary tumor plasmas to high-performance quantitative proteomics analysis. Among the identified proteins, LCAT was selectively expressed in mixed tumor samples. With further MRM and Western blot validation, we discovered that the LCAT protein is an indicator of aggressive mammary tumors, an advanced stage of cancer, possibly highly metastatic. Interestingly, we also found that LCAT is overexpressed in high-grade and lymphnode-positive breast cancer in silico data. We also demonstrated that LCAT is highly expressed in the sera of advanced-stage human breast cancers within the same classification. In conclusion, we identified a possible common plasma protein biomarker, LCAT, that is highly expressed in aggressive human breast cancer and canine mammary tumor.     

Identification of metastasis-associated proteins by proteomic analysis and functional exploration of interleukin-18 in metastasis

Very little is currently known about mechanisms underlying cancer metastasis. In the present study, metastasis-associated proteomes were separated and identified by comparative proteomic analysis, and the metastasis-related function of candidate protein interleukin-18 (IL-18) was further elucidated. First, a pair of highly and poorly metastatic sublines (termed PLA801D and PLA801C, respectively), originating from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach was used to compare the protein expression profiles between PLA801C and PLA801D sublines. Eleven proteins were identified and further verified by one-dimensional Western blotting, Northern blot and/or semiquantitative reverse transciptase polymerase chain reaction analysis. Compared with those in poorly metastatic PLA801C subline, cytokeratin 18, tissue transglutaminase, Rho GDP-dissociation inhibitor 1, tropomyosin, fibroblast type, IL-18 and annexin I were significantly up-regulated, while protein disulfide isomerase, heat shock protein 60, peroxiredoxin 1, chlorine intracellular channel protein 1 (CLI1) and creatine kinase, B chain were significantly down-regulated in the highly metastatic PLA801D subline. Intriguingly, all the identified candidate proteins except for CLI1 have been shown to be somehow associated with distinct aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. Considering that IL-18 was present in highly metastatic PLA801D but absent in poorly metastatic PLA801C, the association of IL-18 with metastasis was further elucidated by introducing IL-18 sense/IL-18 antisense into PLA801C/PLA801D sublines simultaneously. The results demonstrated that ectopically expressed IL-18 promoted cell motility in vitro and down-regulated E-cadherin expression of PLA801C transfectants, while IL-18 antisense remarkably decreased cell invasion potency in vitro and notably increased E-cadherin expression of PLA801D transfectants, indicating that IL-18 might play a role in metastasis by inhibiting E-cadherin expression.     

Phenotype‐rescue of cyclin‐dependent kinase inhibitor p16/INK4A defects in a spontaneous canine cell model of breast cancer

Mammary cancer is among the most frequently observed canine tumors in unspayed female dogs resulting in death due to metastatic disease. These tumors are excellent models of human breast cancer but until recently there was only anecdotal evidence regarding underlying genetic defects. We recently reported expression defects in the cyclin‐dependent kinase p21/Cip1 and p53 among three independent canine mammary tumor (CMT) cell lines derived from spontaneous canine mammary cancers. We investigated further defects in the same three cell lines focusing on additional tumor suppressor gene defects in cyclin‐dependent kinase inhibitors. p27/KIP1 appeared normally expressed and did not appear to encode inactivating mutations. In contrast, expression of p16/INK4A was defective/absent in two cell lines and normal/slightly induced in the third cell line. To determine if defects were causative in maintaining the transformed phenotype, a p16/INK4A transgene was permanently transfected followed by selection and single cell cloning. CMT/p16 clones were characterized for transgene expression, p16 protein content and phenotype including proliferation rate, cell cycle phase distribution, contact inhibition, substrate dependent cell growth and cell morphology. All cell lines appeared unique yet clear indications of phenotype rescue due to p16/INK4A transgene complementation were observed suggesting that defects in p16 expression were present in all three. In some cases cellular senescence also appeared to be induced. These data provide evidence supporting p16/INK4A mutations as causative defects promoting transformation in canine mammary cancer and further characterizes tumor suppressor gene defects with functional consequences in these cells supporting their application as spontaneous animal models of human disease. J. Cell. Biochem. 106: 491–505, 2009. © 2009 Wiley‐Liss, Inc.     

Immunohistochemical and molecular analysis of caveolin-1 expression in canine mammary tumors

Caveolin-1 (Cav-1) is a structural protein present in invaginations of the cell membrane. In human breast cancer, the cav-1 gene is believed to be a tumor suppressor gene associated with inhibition of tumor metastasis. However, little is known about its expression, regulation and function in canine mammary tumors. Expression levels of cav-1 were investigated using real-time PCR and immunohistochemical detection with an anti-human Cav-1 antibody. Gene expression stability of different samples was analyzed using the geNorm software. Mammary tumors from 51 female dogs were compared to normal mammary tissue from 10 female dogs. Malignant mammary cells showed a loss of Cav-1 expression by quantitative RT-PCR and weak Cav-1 staining by immunohistochemistry compared to normal mammary gland tissue. There was a significant relationship between outcome and immunostaining as well as with tumor size, indicating that caveolin subexpression has a positive predictive value and is related to higher survival and smaller tumor size. Our findings indicate that Cav-1 is a potential prognostic marker for canine mammary tumors.     

Proteome analysis of human androgen-independent prostate cancer cell lines: variable metastatic potentials correlated with vimentin expression

To better understand the molecular mechanisms of prostate cancer (PCA) dissemination and to develop new anti-metastasis therapies, key regulatory molecules involved in PCA metastasis were identified in two human androgen-independent PCA cell lines, highly metastatic 1E8-H and lowly metastatic 2B4-L cells. Through 2-DE and MS analyses, 12 proteins with different expression levels in the two cell lines were identified. The following proteins were found to be significantly up-regulated in 1E8-H cells compared with 2B4-L cells: gp96 precursor, calreticulin precursor, vimentin (VIM), Hsp90alpha, peroxiredoxin 2, HNRPH1, ezrin, T-complex protein 1, alpha subunit, and hypothetical protein mln2339. In contrast, heart L-lactate dehydrogenase H chain, annexin I, and protein disulfide isomerase were notably down-regulated in 1E8-H cells compared with 2B4-L cells. To our knowledge, this study is the first to demonstrate that up-regulation of VIM expression positively correlates with the invasion and metastasis of androgen-independent PCA.     

Osteoactivin promotes breast cancer metastasis to bone

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

Bcl-2-mediated cell survival promotes metastasis of EpH4 betaMEKDD mammary epithelial cells

The majority of patients who succumb to cancer die from metastatic disease progression rather than from the primary tumor. Elucidation of the mechanisms underlying tissue-specific metastasis is essential to the development of effective therapies. The mitogen-activated protein kinase kinase (MEK) pathway is frequently activated in human tumors and has been shown to regulate genes involved in proliferation, migration, and invasion. Studies with MEK-transformed EpH4 mouse mammary epithelial cells showed that these cells are highly tumorigenic but have a limited metastatic ability. Detachment of epithelial cells from the extracellular matrix causes disruption of the actin cytoskeleton and induces apoptosis. Several metastatic breast carcinoma cell lines have been shown to be resistant to cell death following actin disruption. This death-resistant phenotype can be modeled by overexpressing the antiapoptotic Bcl-2 protein in cells. This suggests that mechanisms that regulate survival of extravasated tumor cells may enhance metastatic efficiency. Therefore, we examined whether expression of Bcl-2 in MEK-transformed EpH4 mammary epithelial cells could provide a survival advantage and promote metastasis. Expression of Bcl-2 in parental EpH4 mammary epithelial cells or MEK-transformed cells was insufficient to induce increased migration, invasion, or tumor development. However, Bcl-2 expression markedly enhanced spontaneous lung metastasis from orthotopically implanted primary tumors. These results clearly show that mechanisms that regulate primary tumor development are distinct from those that promote metastasis and that assays designed to isolate genes involved in transformation may fail to identify genes that are critical regulators of metastasis.     

Suppression of insulin receptor substrate 1 (IRS-1) promotes mammary tumor metastasis

The insulin receptor substrate (IRS) proteins are cytoplasmic adaptors that organize signaling complexes downstream of activated cell surface receptors. Here, we show that IRS-1 and IRS-2, despite significant homology, play critical yet distinct functions in breast cancer, and we identify specific signaling pathways that are influenced by IRS-1 using the polyoma virus middle-T (PyV-MT) transgenic mouse model of mammary carcinoma and Irs-1 null (Irs1(-/-)) mice. The absence of Irs-1 expression enhanced metastatic spread significantly without a significant effect on primary tumor growth. Orthotopic transplant studies revealed that the increased metastatic potential of Irs1-deficient tumor cells is cell autonomous. Mammary tumors that developed in PyV-MT::Irs1(-/-) mice exhibited elevated Irs-2 function and enhanced phosphatidylinositol 3-kinase/Akt/mTor activity, suggesting that one mechanism by which Irs-1 impedes metastasis is to suppress Irs-2-dependent signaling. In support of this mechanism, reduction of Irs-2 expression in Irs1(-/-) tumor cells restored mTor signaling to wild-type levels. PyV-MT::Irs1(-/-) tumors also exhibited a significant increase in vascular endothelial growth factor expression and microvessel density, which could facilitate their dissemination. The significance of our findings for human breast cancer is heightened by our observation that Irs-1 is inactivated in wild-type, metastatic mammary tumors by serine phosphorylation. Collectively, our findings reveal that inactivation of IRS-1 enhances breast cancer metastasis and support the novel hypothesis that IRS-1 has metastasis suppressor functions for breast cancer.     

The MicroRNA-23b/27b/24 Cluster Promotes Breast Cancer Lung Metastasis by Targeting Metastasis-suppressive Gene Prosaposin

MicroRNAs (miRNAs) have been shown to function as key regulators of tumor progression and metastasis. Recent studies have indicated that the miRNAs comprising the miR-23b/27b/24 cluster might influence tumor metastasis, although the precise nature of this regulation remains unclear. Here, expression of the miR-23b/27b/24 cluster is found to correlate with metastatic potential in mouse and human breast cancer cell lines and is elevated in metastatic lung lesions in human breast cancer patients. Ectopic expression of the miRNAs in the weakly metastatic mouse 4TO7 mammary tumor cell line had no effect on proliferation or morphology of tumor cells in vitro but was found to increase lung metastasis in a mouse model of breast cancer metastasis. Furthermore, gene expression profiling analysis of miRNA overexpressing 4TO7 cells revealed the direct targeting of prosaposin (PSAP), which encodes a secreted protein found to be inversely correlated with metastatic progression in human breast cancer patients. Importantly, ectopic expression of PSAP was able to suppress the metastatic phenotype in highly metastatic 4T1 and MDA-MB-231 SCP28 cells, as well as in cells ectopically expressing miR-23b/27b/24. These findings support a metastasis-promoting function of the miR-23b/27b/24 cluster of miRNAs, which functions in part through the direct inhibition of PSAP.     

Divergent Roles for IRS-1 and IRS-2 in Breast Cancer Metastasis

The insulin receptor substrate (IRS) proteins are cytoplasmic docking proteins that function as essential signaling intermediates downstream of activated cell surface receptors, many of which have been implicated in breast cancer. The IRS proteins do not contain intrinsic kinase activity but rather function by organizing signaling complexes to initiate intracellular signaling cascades. IRS-1 and IRS-2 are expressed in normal mammary epithelial cells and in breast carcinoma cells, where they have been implicated in mediating signals to promote tumor cell survival, growth and motility. Although IRS-1 and IRS-2 are homologous, recent studies have revealed distinct functions for these adaptor proteins in regulating breast cancer progression. Specifically, IRS-2 is a positive regulator of metastasis, whereas IRS-1 may be a suppressor of metastasis. The observation that IRS-1 is inactivated in metastatic mammary tumors raises the possibility that IRS activity, rather than expression, may be a novel predictive indicator of metastasis. Understanding how the IRS proteins function in tumor progression is essential for future efforts aimed at developing approaches to target IRS-1 and IRS-2 in a diagnostic or therapeutic manner for the benefit of breast cancer patients.     

原肌球蛋白、波形纤维蛋白和热休克蛋白70在肝癌转移亚细胞中表达上调

建立并应用人H74 0 2肝癌细胞SCID鼠肿瘤转移模型 ,从转移肺组织经原代细胞培养 ,筛选并建立转移亚细胞系M H74 0 2 ,进而运用蛋白质组学技术筛选肿瘤转移相关蛋白 .通过二维电泳技术检测 ,比较M H74 0 2细胞和亲本H74 0 2细胞的总蛋白 ,从多个差异蛋白质点中选择出 3个在M H74 0 2细胞中表达明显上调的蛋白质点进行ESI QUAD TOF质谱分析 ,并在MSDB公共蛋白数据库中进行同源比较和分析鉴定 .初步确定这 3个蛋白质分别为原肌球蛋白 (tropomyosin) ,波形纤维蛋白 (vimentin)和热休克蛋白 70 (heatshock 70protein ,HSP70 ) .这些蛋白质参与细胞骨架构成、蛋白质折叠和蛋白质相互作用等许多正常生理活动 ,并有报道原肌球蛋白和波形纤维蛋白与肿瘤转移有关 .利用蛋白质组学方法发现 ,在肝癌转移亚细胞M H74 0 2中 ,原肌球蛋白、波形纤维蛋白和热休克蛋白 70表达明显上调 ,进一步揭示它们在肿瘤转移中具有重要的作用 .     

GT1b in human metastatic brain tumors: GT1b as a brain metastasis-associated ganglioside1

We studied ganglioside expression in 12 human metastatic brain tumors metastasized from colon (4), renal (3), lung (2), esophagus (1), pancreas (1), and mammary (1) carcinomas. GM3 was the major common ganglioside expressed in brain metastatic tumor tissues, and GT1b was also present in all the metastatic brain tumor tissues. The latter was identified by TLC-immunostaining and characterized structurally by secondary ion mass spectrometry combined with ‘Far-Eastern blot’. The immunohistochemical analysis of frozen tissue sections confirmed localization of GT1b in the tumor cell membrane or cytosol. GT1b was shown to be expressed both in the primary colon carcinoma and the metastasis of a single patient by immunohistochemical procedure. In systemic carcinomas without brain metastasis, GM3 was a common major component, but no GT1b was detected. These findings indicate that GT1b is a brain metastasis-associated ganglioside. We speculate that the presence of GT1b would be a useful marker for estimating metastatic potentials to the brain.     

Matricellular CCN6 (WISP3) protein: a tumor suppressor for mammary metaplastic carcinomas

Located at 6q22–23, Ccn6 (WISP3) encodes for a matrix-associated protein of the CCN family, characterized by regulatory, rather than structural, roles in development and cancer. CCN6, the least studied member of the CCN family, shares the conserved multimodular structure of CCN proteins, as well as their tissue and cell-type specific functions. In the breast, CCN6 is a critical regulator of epithelial-to-mesenchymal transitions (EMT) and tumor initiating cells. Studies using human breast cancer tissue samples demonstrated that CCN6 messenger RNA and protein are expressed in normal breast epithelia but reduced or lost in aggressive breast cancer phenotypes, especially inflammatory breast cancer and metaplastic carcinomas. Metaplastic carcinomas are mesenchymal-like triple negative breast carcinomas, enriched for markers of EMT and stemness. RNAseq analyses of the TCGA Breast Cancer cohort show reduced CCN6 expression in approximately 50% of metaplastic carcinomas compared to normal breast. Our group identified frameshift mutations of Ccn6 in a subset of human metaplastic breast carcinoma. Importantly, conditional, mammary epithelial-cell specific ccn6 (wisp3) knockout mice develop invasive high-grade mammary carcinomas that recapitulate human spindle cell metaplastic carcinomas, demonstrating a tumor suppressor function for ccn6. Our studies on CCN6 functions in metaplastic carcinoma highlight the potential of CCN6 as a novel therapeutic approach for this specific type of breast cancer.     

Matrix metalloproteinase 13 is induced in fibroblasts in polyomavirus middle T antigen-driven mammary carcinoma without influencing tumor progression

Matrix metalloproteinase (MMP) 13 (collagenase 3) is an extracellular matrix remodeling enzyme that is induced in myofibroblasts during the earliest invasive stages of human breast carcinoma, suggesting that it is involved in tumor progression. During progression of mammary carcinomas in the polyoma virus middle T oncogene mouse model (MMTV-PyMT), Mmp13 mRNA was strongly upregulated concurrently with the transition to invasive and metastatic carcinomas. As in human tumors, Mmp13 mRNA was found in myofibroblasts of invasive grade II and III carcinomas, but not in benign grade I and II mammary intraepithelial neoplasias. To determine if MMP13 plays a role in tumor progression, we crossed MMTV-PyMT mice with Mmp13 deficient mice. The absence of MMP13 did not influence tumor growth, vascularization, progression to more advanced tumor stages, or metastasis to the lungs, and the absence of MMP13 was not compensated for by expression of other MMPs or tissue inhibitor of metalloproteinases. However, an increased fraction of thin collagen fibrils was identified in MMTV-PyMT;Mmp13(-/-) compared to MMTV-PyMT;Mmp13(+/+) tumors, showing that collagen metabolism was altered in the absence of MMP13. We conclude that the expression pattern of Mmp13 mRNA in myofibroblasts of invasive carcinomas in the MMTV-PyMT breast cancer model recapitulates the expression pattern observed in human breast cancer. Our results suggest that MMP13 is a marker of carcinoma-associated myofibroblasts of invasive carcinoma, even though it does not make a major contribution to tumor progression in the MMTV-PyMT breast cancer model.     

Molecular Plasticity of E-Cadherin and Sialyl Lewis X Expression,in Two Comparative Models of Mammary Tumorigenesis

Background

The process of metastasis involves a series of steps and interactions between the tumor embolus and the microenvironment. Key alterations in adhesion molecules are known to dictate progression from the invasive to malignant phenotype followed by colonization at a distant site. The invasive phenotype results from the loss of expression of the E-cadherin adhesion molecule, whereas the malignant phenotype is associated with an increased expression of the carbohydrate ligand-binding epitopes, (e.g. Sialyl Lewis ^x/a) that bind endothelial E-selectin of the lymphatics and vasculature.

Methodology

Our study analyzed the expression of two adhesion molecules, E-cadherin and Sialyl Lewis x (sLe^x), in both a canine mammary carcinoma and human inflammatory breast cancer (IBC) model, using double labelled immunofluorescence staining.

Results

Our results demonstrate that canine mammary carcinoma and human IBC exhibit an inversely correlated cellular expression of E-cadherin and sLe^x within the same tumor embolus.

Conclusions

Our results in these two comparative models (canine and human) suggest the existence of a biologically coordinated mechanism of E-cadherin and sLe^x expression (i.e. molecular plasticity) essential for tumor establishment and metastatic progression.