Osteopontin induces multiple changes in gene expression that reflect the six "hallmarks of cancer" in a model of breast cancer progression

Tumor progression is a multistep process, which enables cells to evolve from benign to malignant tumors. This progression has been suggested to depend on six essential characteristics identified as the "hallmarks of cancer," which include: self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. Osteopontin (OPN) is an integrin-binding protein that has been shown to be associated with the progression of several cancer types, and to play an important functional role in various aspects of malignancy, particularly tissue invasion and metastasis. Here we studied genes regulated by OPN in a model of human breast cancer using oligonucleotide microarray technology by comparing the gene-expression profiles of 21NT mammary carcinoma cells transfected to overexpress OPN versus mock-transfected control cells. From over 12,000 human genes, we identified 99 known human genes differentially regulated by OPN whose expression changed by at least 1.5-fold and showed statistically significant differences in mean expression levels between groups. Functional classification of these genes into the hallmarks of cancer categories showed that OPN can affect the expression of genes involved in all six categories in this model. Furthermore, we were able to validate the expression of 18/19 selected candidate genes by quantitative real-time PCR, further supporting our microarray findings. This study provides the first evidence that OPN can lead to numerous gene expression changes that influence multiple aspects of tumor progression and malignant growth.     

Inhibition of invasion and metastasis by glypican-3 in a syngeneic breast cancer model

Glypican-3 (GPC3), a proteoglycan bound to the cell membrane through a GPI anchor, is widely expressed in the embryo but down regulated in most adult tissues, with some exceptions as mammary cells. GPC3 is involved in the regulation of cell proliferation and survival in specific cell types. LM3, a murine mammary tumor cell line unable to express GPC3, was stably transfected with the rat GPC3 gene to analyze its role in tumor progression. Upon injection into syngeneic BALB/c mice LM3-GPC3 clones showed less local invasiveness and developed fewer spontaneous and experimental lung metastasis than controls. GPC3-expressing cells were more sensitive to apoptosis induced by serum depletion, exhibited a delay in the first steps of spreading and were less motile than controls. On the other hand, LM3-GPC3 cells were significantly more adherent to FN than control ones. We observed that GPC3 transfectants presented a higher expression of E-cadherin and -catenin, molecules whose down regulation has been associated with tumor progression. Exogenous TGF- increased MMP-9 activity in both control and GPC3-expressing cells, but did not modulate MMP-2. Contrarily, GPC3 expression prevented the increase of MMP-2 activity induced by IGF-II. Our results suggest that GPC3 has a protective role against mammary cancer progression.     

Cancer‐associated fibroblast‐derived Lumican promotes gastric cancer progression via the integrin β1‐FAK signaling pathway

Gastric cancer (GC) is one of the most frequent malignant tumors worldwide and is associated with high invasiveness, high metastasis and poor prognosis. Cancer‐associated fibroblasts (CAFs), residing around tumor cells in tumor stroma, are important modifiers of tumor initiation and progression. However, the molecular mechanisms by which CAF's modulate tumor development have yet not to be characterized in GC. Here we performed tissue assay analyses identifying that Lumican, an extracellular matrix protein, is highly expressed in human gastric CAFs and its expression is positively associated with depth of invasion, lymph node metastasis, TNM stage and poor survival rate of GC. Functional studies revealed that integrin β1‐FAK signaling pathways mediate the promoting effect of Lumican on GC cell proliferation, migration and invasion in vitro. In accordance with these observations, in GC cells co‐cultured with CAFs in which Lumican had been knocked down, decreased gastric tumor growth and metastasis in vivo was apparent. In summary, CAF‐derived Lumican contributes to tumorigenesis and metastasis of GC by activating the integrin β1‐FAK signaling pathway.     

Stromal expression of ALDH1 in human breast carcinomas indicates reduced tumor progression

Interactions between cancer cells and microenvironment are emerging issue in tumor progression. Aldehyde dehydrogenase 1 (ALDH1) is a recognized cancer stem cell marker but little is known about its role in intratumoral stroma. Therefore, we focused on ALDH1 expression in tumor-associated stroma of breast carcinomas (BrCa).Stromal and tumoral ALDH1 expression was evaluated immunohistochemically in BrCa and their lymph node metastases (LNMs), and related to clinico-pathological characteristics, patients’ outcome, presence of CD68, HLADR, retinoic acid (RA) in stroma, and selected proteins in tumor cells.ALDH1(+) stromal cells were detected in 53% of 374 BrCa and 61% of 102 LNMs. ALDH1(+) stroma in primary tumor correlated to longer disease-free (p = 0.030), metastasis-free (p = 0.024), and overall survival (p = 0.043) having an independent prognostic impact on DFS (multivariate analysis, p = 0.047). It was associated with concomitant presence of HLA-DR(+) stromal cells and RA in tumor cells (both p < 0.001), and inversely associated with vimentin expression in tumor cells (p = 0.036). ALDH1(+) stroma in LNMs correlated inversely to presence of disseminated tumor cells in patients’ bone marrow (p = 0.014) and was independent prognosticator of shorter DFS and MFS (multivariate analysis, p = 0.004 and p = 0.002, respectively).In conclusion, ALDH1 expression in tumor-associated stromal cells indicates reduced BrCa progression, possibly via RA secretion.     

Serial transplantation of NMU-induced rat mammary tumors: a model of human breast cancer progression

Human breast cancer is a heterogeneous disease that appears to progress from an in situ tumor to invasive cancer. Little is known about the molecular events driving this progression. Although microarray technology has helped us understand the genetic heterogeneity of breast cancer, its application to studying the transition from in situ to invasive disease is limited by the inability to follow the progression of a single patient's tumor. We previously used rat specific microarrays to show that N-methyl-N-nitrosourea induced tumors are similar to low-grade estrogen-receptor positive human breast cancer. Here, we transplanted these tumors through 5 generations of syngeneic hosts, and studied 65 resulting tumors. Most transplanted tumors gradually progressed from a noninvasive, low-grade cancer to a higher-grade invasive disease, losing p63 localization and basement membrane integrity. Invasive cancers frequently demonstrated a more mesenchymal phenotype with increased vimentin expression. Additionally, a unique transplant series is described with a phenotype similar to human basal-like breast cancer. Rat-specific Affymetrix gene arrays containing 15,866 gene probes identified genes that differentiated highly invasive tumors from those of low invasive potential. A linear regression analysis was used to find genes whose change in expression paralleled increasing invasive features independent of the transplant lineage of origin. Genes identified were assigned membership in cell adhesion, signal transduction, cell cycle and extracellular matrix groups, among others. This animal model overcomes the difficulty in studying human breast cancer progression. Our data support a gradual and continuous alteration in programs of gene expression during breast cancer invasion.     

Expression of mucin 1 possessing a 3′‐sulfated core1 in recurrent and metastatic breast cancer

Breast cancer is the most frequent cancer threatening the lives of women between the ages of 30 and 64. The cancer antigen 15‐3 assay (CA15‐3) has been widely used for the detection of breast cancer recurrence; however, its sensitivity and specificity are inadequate. We previously found that the breast cancer cell line YMBS secretes mucin 1 possessing 3′‐sulfated core1 (3Score1‐MUC1) into the medium. Therefore, we here evaluated whether 3Score1‐MUC1 is secreted into the blood streams of breast cancer patients, and whether it can serve as an improved breast cancer marker. We developed a lectin‐sandwich immunoassay, called Gal4/MUC1, using a 3′‐sulfated core1‐specific galectin‐4 and a MUC1 monoclonal antibody. Using the Gal4/MUC1 assay method, we found that 3Score1‐MUC1 was profoundly expressed in the blood streams of patients with recurrent and/or metastatic breast cancer. The positive ratio of the Gal4/MUC1 assay was higher than that of the CA15‐3 assay in both primary (n = 240) and relapsed (n = 43) patients, especially in the latter of which the positive ratio of Gal4/MUC1 was 86%. whereas that of CA15‐3 was 47%. Furthermore, serum Gal4/MUC1 levels could more sensitively reflect the recurrence of primary breast cancer patients after surgery. Therefore, the Gal4/MUC1 assay should be an excellent alternative to the CA15‐3 tumor marker for tracking the recurrence and metastasis of breast cancer.     

p53/Lactate dehydrogenase A axis negatively regulates aerobic glycolysis and tumor progression in breast cancer expressing wild‐type p53

Tumor suppressor p53 is a master regulator of apoptosis and plays key roles in cell cycle checkpoints. p53 responds to metabolic changes and alters metabolism through several mechanisms in cancer. Lactate dehydrogenase A (LDHA), a key enzyme in glycolysis, is highly expressed in a variety of tumors and catalyzes pyruvate to lactate. In the present study, we first analyzed the association and clinical significance of p53 and LDHA in breast cancer expressing wild‐type p53 (wt‐p53) and found that LDHA mRNA levels are negatively correlated with wt‐p53 but not with mutation p53 mRNA levels, and low p53 and high LDHA expression are significantly associated with poor overall survival rates. Furthermore, p53 negatively regulates LDHA expression by directly binding its promoter region. Moreover, a series of LDHA gain‐of‐function and rescore experiments were carried out in breast cancer MCF7 cells expressing endogenous wt‐p53, showing that ectopic expression of p53 decreases aerobic glycolysis, cell proliferation, migration, invasion and tumor formation of breast cancer cells and that restoration of the expression of LDHA in p53‐overexpressing cells could abolish the suppressive effect of p53 on aerobic glycolysis and other malignant phenotypes. In conclusion, our findings showed that repression of LDHA induced by wt‐p53 blocks tumor growth and invasion through downregulation of aerobic glycolysis in breast cancer, providing new insights into the mechanism by which p53 contributes to the development and progression of breast cancer.     

Gastric cancer progression may involve a shift in HLA‐E profile from an intact heterodimer to β2‐microglobulin‐free monomer

Phenotypic expression of human leukocyte antigen (HLA)‐E on the surface of tumor lesions includes intact heterodimer [HLA‐E heavy chain and β2‐microglobulin (β2m)] and β2m‐free monomer. Anti‐HLA‐E monoclonal antibodies (mAbs), MEM‐E/02 or 3D12 bind to the peptide sequences in β2m‐free HLA‐E, which is common and shared with HLA‐Ia monomers. A newly developed monospecific anti‐HLA‐E mAb (TFL‐033) recognizes HLA‐E‐restricted peptide sequences on α1 and α2 helices away from β2‐m‐site. Tumor progression may involve shedding of β2‐m from HLA‐E or overexpression of β2m‐free monomers. There is a need to identify and distinguish the different phenotypic expression of HLA‐E, particularly the intact heterodimer from the β2m‐free monomer on the surface of tumor lesions. Because of the unique peptide‐binding affinities of the mAbs, it is hypothesized that TFL‐033 and MEM‐E/02 may distinguish the phenotypic expressions of cell surface HLA‐E during stages of tumor progression. Only TFL‐033 stained diffusely the cytoplasm of normal mucosa. The incidence and intensity of TFL‐033 staining of the cell surface in early stages, poorly or undifferentiated and non‐nodal lesions and in diffuse carcinoma is greater than that of MEM‐E/02. Whereas MEM‐E/02 stained terminal stages, adenocarcinoma and lymph node metastatic lesions intensely, either owing to increased expression of β2m‐free HLA‐E with tumor progression or owing to expression of HLA‐Ia molecules. Our study evaluates the relative diagnostic potential of HLA‐E‐monospecific TFL‐033 and the HLA‐Ia‐reactive MEM‐E/02 for determining the specific distribution and immunodiagnosis of different phenotypic expression HLA‐E in tumor lesions, and the structural and functional alterations undergone by HLA‐E during tumor progression.     

Serum deprivation response inhibits breast cancer progression by blocking transforming growth factor‐β signaling

Serum deprivation response (SDPR), a key substrate for protein kinase C, play a critical role in inducing membrane curvature and participate in the formation of caveolae. However, the function of SDPR in cancer development and progression is still not clear. Here, we found that SDPR is downregulated in human breast cancer. Overexpression of SDPR suppresses cell proliferation and invasion in MDA‐MB‐231 cells, while depletion of SDPR promotes cell proliferation and invasion in MCF10A cells. Subsequently, SDPR depletion induces epithelial–mesenchymal transition (EMT)‐like phenotype. Finally, knockdown of SDPR activates transforming growth factor‐β (TGF‐β) signaling by upregulation of TGF‐β1 expression. In conclusion, our results showed that SDPR inhibits breast cancer progression by blocking TGF‐β signaling. Serum deprivation response suppresses cell proliferation and invasion in breast cancer cells. SDPR depletion induces epithelial–mesenchymal transition by activation of TGF‐β signaling.     

Fear of cancer progression and cancer‐related intrusive cognitions in breast cancer survivors

Objective: To assess the character and frequency of fear of progression (FoP) and to clarify its relationship with cancer‐related intrusive cognitions in breast cancer survivors. Methods: A sample of 1083 patients was recruited in this cross‐sectional study through a population‐based Cancer Registry an average of 47 month following diagnosis (66% response rate). Participants completed self‐report measures assessing fear of cancer progression (FoP‐Q‐SF), posttraumatic stress‐disorder symptoms (PCL‐C), coping strategies (DWI) and quality of life (QoL) (SF‐8). Results: In total, 23.6% of women were classified as having moderate to high FoP. Being nervous prior to doctors' appointments or examinations and being afraid of relying on strangers for activities of daily living were the most frequent fears. FoP was significantly associated with younger age, having children, disease progress, chemotherapy, perceived amount of impairments, physical and mental QoL, but not with time since initial diagnosis. Intrusive cognitions were screened in 37% of the sample. We found significant correlations between FoP and intrusive thoughts (r=0.63), avoidance (r=0.57), hyperarousal (r=0.54) and posttraumatic stress disorder diagnosis (r=0.42). Factors significantly associated with moderate and high FoP included a depressive coping style as well as an active problem‐oriented coping style, intrusion, avoidance and hyperarousal symptoms (Nagelkerke's R²=0.44). Conclusions: Findings of this study give information regarding the frequency and the character of anxiety in breast cancer survivors and underline the relation of FoP to the reality of living with breast cancer. Results suggest that intrusive cognitions as well as avoidance and hyperarousal symptoms seem to be closely related to future‐oriented fears of cancer recurrence. Copyright © 2009 John Wiley & Sons, Ltd.     

Stromal immunoglobulin κC expression is associated with initiation of breast cancer in TA2 mice and human breast cancer

The initiation of spontaneous breast cancer (SBC) in Tientsin Albino 2 (TA2) mice is related to mouse mammary tumor virus (MMTV) infection, and MMTV amplification is hormonally regulated. To explore the insertion site of MMTVLTR in TA2 mouse genome, reverse PCR and nested PCR were used to amplify the unknown sequence on both sides of the MMTV‐LTRSAG gene in SBC and normal breast tissue of TA2 mice. Furthermore, the clinicopathological significance of the insertion site was evaluated in 43 samples of normal breast tissue, 46 samples of breast cystic hyperplasia, 54 samples of ductal carcinoma in situ, 142 samples of primary breast cancer and 47 samples of lymph node metastatic breast cancer by RNA in situ hybridization. We confirmed that the insertion site of the MMTV‐LTRSAG gene was located between Ig κ v2‐112 and Ig κ v14‐111 in chromosome 6 of TA2 mouse. IG κ C was localized in the stromal cells of TA2 mouse with SBC and in human breast cancer tissues. Tumor cells were negative for IG κ C in RNA in situ hybridization. The positive staining index of IG κ C in stromal cells was the highest in lymph node metastatic breast cancer, followed by primary breast cancer, ductal carcinoma in situ, and breast cystic hyperplasia. Furthermore, the positive staining index of IG κ C was related to the expression of ER, PR, HER2 and Ki‐67. Our findings showed that stromal IG κ C expression was associated with the initiation of SBC in TA2 mice. IG κ C may be a high‐risk factor for the initiation and progression of human breast cancer.     

Breast cancer‐derived transforming growth factor‐β and tumor necrosis factor‐α compromise interferon‐α production by tumor‐associated plasmacytoid dendritic cells

We previously reported that plasmacytoid dendritic cells (pDCs) infiltrating breast tumors are impaired for their interferon‐α (IFN‐α) production, resulting in local regulatory T cells amplification. We designed our study to decipher molecular mechanisms of such functional defect of tumor‐associated pDC (TApDC) in breast cancer. We demonstrate that besides IFN‐α, the production by Toll‐like receptor (TLR)‐activated healthy pDC of IFN‐β and TNF‐α but not IP‐10/CXCL10 nor MIP1‐α/CCL3 is impaired by the breast tumor environment. Importantly, we identified TGF‐β and TNF‐α as major soluble factors involved in TApDC functional alteration. Indeed, recombinant TGF‐β1 and TNF‐α synergistically blocked IFN‐α production of TLR‐activated pDC, and neutralization of TGF‐β and TNF‐α in tumor‐derived supernatants restored pDCs' IFN‐α production. The involvment of tumor‐derived TGF‐β was further confirmed in situ by the detection of phosphorylated Smad2 in the nuclei of TApDC in breast tumor tissues. Mechanisms of type I IFN inhibition did not involve TLR downregulation but the inhibition of IRF‐7 expression and nuclear translocation in pDC after their exposure to tumor‐derived supernatants or recombinant TGF‐β1 and TNF‐α. Our findings indicate that targeting TApDC to restore their IFN‐α production might be an achievable strategy to induce antitumor immunity in breast cancer by combining TLR7/9‐based immunotherapy with TGF‐β and TNF‐α antagonists.