Proline-Rich Homeodomain protein (PRH/HHEX) is a suppressor of breast tumour growth.
ABSTRACT: Breast tumours progress from hyperplasia to ductal carcinoma in situ (DCIS) and invasive breast carcinoma (IBC). PRH/HHEX (proline-rich homeodomain/haematopoietically expressed homeobox) is a transcription factor that displays both tumour suppressor and oncogenic activity in different disease contexts; however, the role of PRH in breast cancer is poorly understood. Here we show that nuclear localization of the PRH protein is decreased in DCIS and IBC compared with normal breast. Our previous work has shown that PRH phosphorylation by protein kinase CK2 prevents PRH from binding to DNA and regulating the transcription of multiple genes encoding growth factors and growth factor receptors. Here we show that transcriptionally inactive phosphorylated PRH is elevated in DCIS and IBC compared with normal breast. To determine the consequences of PRH loss of function in breast cancer cells, we generated inducible PRH depletion in MCF-7 cells. We show that PRH depletion results in increased MCF-7 cell proliferation in part at least due to increased vascular endothelial growth factor signalling. Moreover, we demonstrate that PRH depletion increases the formation of breast cancer cells with cancer stem cell-like properties. Finally, and in keeping with these findings, we show that PRH overexpression inhibits the growth of mammary tumours in mice. Collectively, these data indicate that PRH plays a tumour suppressive role in the breast and they provide an explanation for the finding that low PRH mRNA levels are associated with a poor prognosis in breast cancer.
Project description:Breast tumours progress from hyperplasia to ductal carcinoma in situ (DCIS) and invasive breast carcinoma (IBC). PRH/HHEX (Proline Rich Homeodomain/Haematopoietically expressed homeobox) is a transcription factor that displays both tumour suppressor and oncogenic activity in different disease contexts however, the role of PRH in breast cancer is poorly understood. To determine the consequences of PRH loss of function in breast cancer cells we generated inducible PRH depletion in MCF-7 cells. We show that PRH depletion results in increased MCF-7 cell proliferation in part at least due to increased vascular endothelial growth factor signaling. Moreover we demonstrate that PRH depletion increases the formation of breast cancer cells with cancer stem cell-like properties. Finally, and in keeping with these findings, we show that PRH over-expression inhibits the growth of mammary tumours in mice. Collectively these data indicate that PRH plays a tumour suppressive role in the breast and they provide an explanation for the finding that low PRH mRNA levels are associated with a poor prognosis in breast cancer. Overall design: A 12 array study in which Proline-Rich Homeodomain protein (PRH/HHEX) is either knocked down or overexpressed in MCF7 cells. In the knockdown experiment there are 3 scrambled shRNA control replicates and 3 PRH shRNA replicates. In the overexpression experiment there are 3 empty vector transfected control replicates and 3 PRH transfected overexpression replicates.
Project description:BACKGROUND:The transition from ductal carcinoma in situ (DCIS) to invasive breast carcinoma (IBC) is an important step during breast carcinogenesis. Understanding its molecular changes may help to identify high-risk DCIS that progress to IBC. Here, we describe a transcriptomic profiling analysis of matched formalin-fixed and paraffin-embedded (FFPE) DCIS and IBC components of individual breast tumours, containing both tumour compartments. The study was performed to validate progression-associated transcripts detected in an earlier gene profiling project using fresh frozen breast cancer tissue. In addition, FFPE tissues from patients with pure DCIS (pDCIS) were analysed to identify candidate transcripts characterizing DCIS with a high or low risk of progressing to IBC. METHODS:Fifteen laser microdissected pairs of DCIS and IBC were profiled by Illumina DASL technology and used for expression validation by qPCR. Differential expression was independently validated using further 25 laser microdissected DCIS/IBC sample pairs. Additionally, laser microdissected epithelial cells from 31 pDCIS were investigated for expression of candidate transcripts using qPCR. RESULTS:Multiple statistical calculation methods revealed 1784 mRNAs which are differentially expressed between DCIS and IBC (P < 0.05), of which 124 have also been identified in the gene profiling project using fresh frozen breast cancer tissue. Nine mRNAs that had been selected from the gene list obtained using fresh frozen tissues by applying pathway and network analysis (MMP11, GREM1, PLEKHC1, SULF1, THBS2, CSPG2, COL10A1, COL11A1, KRT14) were investigated in tissues from the same 15 microdissected specimens and the 25 independent tissue samples by qPCR. All selected transcripts were also detected in tumour cells from pDCIS. Expression of MMP11 and COL10A1 increased significantly from pDCIS to DCIS of DCIS/IBC mixed tumours. CONCLUSION:We confirm differential expression of progression-associated transcripts in FFPE breast cancer samples which might mediate the transition from DCIS to IBC. MMP11 and COL10A1 may characterize pure DCIS with a high risk developing IDC.
Project description:Cyclooxygenase type-2 (COX-2) is overexpressed in malignant tumours including breast cancers, though the mechanism of upregulation is unclear. This study aimed to determine COX-2 expression in ductal carcinoma in situ (DCIS) in comparison to invasive breast cancer (IBC) and normal breast, and also to investigate the relationship of COX-2 expression with HER-2 expression, oestrogen receptor (ER), tumour grade and cellular proliferation (Ki67) in DCIS. Cyclooxygenase type-2, HER-2, ER and Ki67 expression were determined by immunohistochemistry on paraffin tissue sections of DCIS (n=187), IBC (n=65) and normal breast reduction tissue (n=60). Cyclooxygenase type-2 expression in DCIS (67%, P<0.001) and IBC (63%, P<0.001) was significantly greater than in normal breast (23%). There was no difference in COX-2 expression level between DCIS and IBC (P=0.87) or between normal breast from reduction mammoplasty tissue and normal breast ducts around DCIS (22%, P=0.29). In DCIS, COX-2 expression was associated with higher cellular proliferation rates (P<0.0001), nuclear grade (P=0.003), with ER negativity (P=0.003) and with HER-2 positivity (P<0.0001). Cyclooxygenase type-2 expression is upregulated in in situ breast cancer and is associated with surrogate markers of an aggressive DCIS phenotype including nonoestrogen-regulated signalling pathways. Cyclooxygenase type-2 inhibition may potentially prevent the development of ER-positive and ER-negative breast cancers.
Project description:Cancer cells go through a process known as epithelial-mesenchymal transition (EMT) during which they acquire the ability to migrate and invade extracellular matrix. Some cells also acquire the ability to move across a layer of endothelial cells to enter and exit the bloodstream; intra- and extravasation, respectively. The transcription factor PRH/HHEX (proline-rich homeodomain/haematopoietically expressed homeobox) controls cell proliferation and cell migration/invasion in a range of cell types. Our previous work showed that PRH activity is downregulated in prostate cancer cells owing to increased inhibitory PRH phosphorylation and that this increases cell proliferation and invasion. PRH inhibits migration and invasion by prostate and breast epithelial cells in part by activating the transcription of Endoglin, a transforming growth factor ? (TGF?) co-receptor. Here we show that depletion of PRH in immortalised prostate epithelial cells results in increased extravasation in vitro. We show that blood platelets stimulate extravasation of cells with depleted PRH and that inhibition of TGF? signalling blocks the effects of platelets on these cells. Moreover, TGF? induces changes characteristic of EMT including decreased E-Cadherin expression and increased Snail expression. We show that in prostate cells PRH regulates multiple genes involved in EMT and TGF? signalling. However, both platelets and TGF? increase PRH phosphorylation. In addition, TGF? increases binding of its effector pSMAD3 to the PRH/HHEX promoter and downregulates PRH protein and mRNA levels. Thus, TGF? signalling downregulates PRH activity by multiple mechanisms and induces an EMT that facilitates extravasation and sensitises cells to TGF?.
Project description:The underlying mechanism of the progression of ductal carcinoma in situ (DCIS), a non-obligate precursor of invasive breast cancer (IBC), has yet to be elucidated. In IBC, Apolipoprotein B mRNA Editing Enzyme, Catalytic Polypeptide-Like 3B (APOBEC3B) is upregulated in a substantial proportion of cases and is associated with higher mutational load and poor prognosis. However, APOBEC3B expression has never been studied in DCIS. We performed mRNA expression analysis of APOBEC3B in synchronous DCIS and IBC and surrounding normal cells. RNA was obtained from 53 patients. The tumors were categorized based on estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (Her2) and phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) mutation status. APOBEC3B mRNA levels were measured by RT-qPCR. The expression levels of paired DCIS and adjacent IBC were compared, including subgroup analyses. The normal cells expressed the lowest levels of APOBEC3B. No differences in expression were found between DCIS and IBC. Subgroup analysis showed that APOBEC3B was the highest in the ER subgroups of DCIS and IBC. While there was no difference in APOBEC3B between wild-type versus mutated PIK3CA DCIS, APOBEC3B was higher in wild-type versus PIK3CA-mutated IBC. In summary, our data show that APOBEC3B is already upregulated in DCIS. This suggests that APOBEC3B could already play a role in early carcinogenesis. Since APOBEC3B is a gain-of-function mutagenic enzyme, patients could benefit from the therapeutic targeting of APOBEC3B in the early non-invasive stage of breast cancer.
Project description:Ductal carcinoma in situ (DCIS) is a non-invasive type of breast cancer with highly variable potential of becoming invasive and affecting mortality. Currently, many patients with DCIS are overtreated due to the lack of specific biomarkers that distinguish low risk lesions from those with a higher risk of progression. In this study, we analyzed 57 pure DCIS and 313 invasive breast cancers (IBC) from different patients. Three levels of genomic data were obtained; gene expression, DNA methylation, and DNA copy number. We performed subtype stratified analyses and identified key differences between DCIS and IBC that suggest subtype specific progression. Prominent differences were found in tumors of the basal-like subtype: Basal-like DCIS were less proliferative and showed a higher degree of differentiation than basal-like IBC. Also, core basal tumors (characterized by high correlation to the basal-like centroid) were not identified amongst DCIS as opposed to IBC. At the copy number level, basal-like DCIS exhibited fewer copy number aberrations compared with basal-like IBC. An intriguing finding through analysis of the methylome was hypermethylation of multiple protocadherin genes in basal-like IBC compared with basal-like DCIS and normal tissue, possibly caused by long range epigenetic silencing. This points to silencing of cell adhesion-related genes specifically in IBC of the basal-like subtype. Our work confirms that subtype stratification is essential when studying progression from DCIS to IBC, and we provide evidence that basal-like DCIS show less aggressive characteristics and question the assumption that basal-like DCIS is a direct precursor of basal-like invasive breast cancer.
Project description:Objectives:Breast malignancy is a serious threat to women's health around the world. Following the rapid progress in the field of cancer diagnostics and identification of pathological markers, breast tumor treatment methods have been greatly improved. However, for invasive, ductal carcinomas and mammary fibroadenoma, there is an urgent demand for better breast tumor-linked biomarkers. The current study was designed to identify diagnostic and/or therapeutic protein biomarkers for breast tumors. Methods:A total of 140 individuals were included, comprising 35 healthy women, 35 invasive breast cancers (IBC), 35 breast ductal carcinomas in situ (DCIS), and 35 breast fibroadenoma patients. Isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis was employed to characterize differentially expressed proteins for potential biomarkers in IBC, DCIS, and fibroadenomas by comparisons with their matched adjacent tissues and/or normal breast tissues. The public databases Metascape and String were used for bioinformatic analyses. Results:Using the proteomics approach, we identified differentially expressed proteins in tissues of different breast tumors compared to normal/adjacent breast tissues, including 100 in IBC, 52 in DCIS, and 44 in fibroadenoma. Among the 100 IBC differentially expressed proteins, 37 were found to be specific to this type of cancer only. Additionally, four proteins were specifically expressed in DCIS and four in fibroadenoma. Compared to corresponding adjacent tissues and normal breast tissues, 18 step-changing proteins were differentially expressed in IBC, 14 in DCIS, and 13 in fibroadenoma, respectively. Compared to DCIS and normal breast tissues, 65 proteins were differentially expressed in IBC with growing levels of malignancy. Conclusions:The identified potential protein biomarkers may be used as diagnostic and/or therapeutic targets in breast tumors.
Project description:PURPOSE:The detection rate of breast ductal carcinoma in situ (DCIS) has increased significantly, raising the concern that DCIS is overdiagnosed and overtreated. Therefore, there is an unmet clinical need to better predict the risk of progression among DCIS patients. Our hypothesis is that by combining molecular signatures with clinicopathologic features, we can elucidate the biology of breast cancer progression, and risk-stratify patients with DCIS. METHODS:Targeted exon sequencing with a custom panel of 223 genes/regions was performed for 125 DCIS cases. Among them, 60 were from cases having concurrent or subsequent invasive breast cancer (IBC) (DCIS?+?IBC group), and 65 from cases with no IBC development over a median follow-up of 13 years (DCIS-only group). Copy number alterations in chromosome 1q32, 8q24, and 11q13 were analyzed using fluorescence in situ hybridization (FISH). Multivariable logistic regression models were fit to the outcome of DCIS progression to IBC as functions of demographic and clinical features. RESULTS:We observed recurrent variants of known IBC-related mutations, and the most commonly mutated genes in DCIS were PIK3CA (34.4%) and TP53 (18.4%). There was an inverse association between PIK3CA kinase domain mutations and progression (Odds Ratio [OR] 10.2, p?<?0.05). Copy number variations in 1q32 and 8q24 were associated with progression (OR 9.3 and 46, respectively; both p?<?0.05). CONCLUSIONS:PIK3CA kinase domain mutations and the absence of copy number gains in DCIS are protective against progression to IBC. These results may guide efforts to distinguish low-risk from high-risk DCIS.
Project description:BACKGROUND:The incidence of ductal carcinoma in situ (DCIS) has increased substantially since the introduction of mammography screening. Nevertheless, little is known about the natural history of preclinical DCIS in the absence of biopsy or complete excision. METHODS:Two well-established population models evaluated six possible DCIS natural history submodels. The submodels assumed 30%, 50%, or 80% of breast lesions progress from undetectable DCIS to preclinical screen-detectable DCIS; each model additionally allowed or prohibited DCIS regression. Preclinical screen-detectable DCIS could also progress to clinical DCIS or invasive breast cancer (IBC). Applying US population screening dissemination patterns, the models projected age-specific DCIS and IBC incidence that were compared to Surveillance, Epidemiology, and End Results data. Models estimated mean sojourn time (MST) in the preclinical screen-detectable DCIS state, overdiagnosis, and the risk of progression from preclinical screen-detectable DCIS. RESULTS:Without biopsy and surgical excision, the majority of DCIS (64-100%) in the preclinical screen-detectable state progressed to IBC in submodels assuming no DCIS regression (36-100% in submodels allowing for DCIS regression). DCIS overdiagnosis differed substantially between models and submodels, 3.1-65.8%. IBC overdiagnosis ranged 1.3-2.4%. Submodels assuming DCIS regression resulted in a higher DCIS overdiagnosis than submodels without DCIS regression. MST for progressive DCIS varied between 0.2 and 2.5?years. CONCLUSIONS:Our findings suggest that the majority of screen-detectable but unbiopsied preclinical DCIS lesions progress to IBC and that the MST is relatively short. Nevertheless, due to the heterogeneity of DCIS, more research is needed to understand the progression of DCIS by grades and molecular subtypes.
Project description:The changes in DNA methylation status in cancer cells are characterized by hypermethylation of promoter CpG islands and diffuse genomic hypomethylation. Alu and long interspersed nucleotide element-1 (LINE-1) are non-coding genomic repetitive sequences and methylation of these elements can be used as a surrogate marker for genome-wide methylation status. This study was designed to evaluate the changes of Alu and LINE-1 hypomethylation during breast cancer progression from normal to pre-invasive lesions and invasive breast cancer (IBC), and their relationship with characteristics of IBC. We analyzed the methylation status of Alu and LINE-1 in 145 cases of breast samples including normal breast tissue, atypical ductal hyperplasia/flat epithelial atypia (ADH/FEA), ductal carcinoma in situ (DCIS) and IBC, and another set of 129 cases of IBC by pyrosequencing. Alu methylation showed no significant changes during multistep progression of breast cancer, although it tended to decrease during the transition from DCIS to IBC. In contrast, LINE-1 methylation significantly decreased from normal to ADH/FEA, while it was similar in ADH/FEA, DCIS and IBC. In IBC, Alu hypomethylation correlated with negative estrogen receptor (ER) status, and LINE-1 hypomethylation was associated with negative ER status, ERBB2 (HER2) amplification and p53 overexpression. Alu and LINE-1 methylation status was significantly different between breast cancer subtypes, and the HER2 enriched subtype had lowest methylation levels. In survival analyses, low Alu methylation status tended to be associated with poor disease-free survival of the patients. Our findings suggest that LINE-1 hypomethylation is an early event and Alu hypomethylation is probably a late event during breast cancer progression, and prominent hypomethylation of Alu and LINE-1 in HER2 enriched subtype may be related to chromosomal instability of this specific subtype.