Jan Bouchal

Novel markers for differentiation of lobular and ductal invasive breast carcinomas by laser microdissection and microarray analysis

BackgroundInvasive ductal and lobular carcinomas (IDC and ILC) are the most common histological types of breast cancer. Clinical follow-up data and metastatic patterns suggest that the development and progression of these tumors are different. The aim of our study was to identify gene expression profiles of IDC and ILC in relation to normal breast epithelial cells.MethodsWe examined 30 samples (normal ductal and lobular cells from 10 patients, IDC cells from 5 patients, ILC cells from 5 patients) microdissected from cryosections of ten mastectomy specimens from postmenopausal patients. Fifty nanograms of total RNA were amplified and labeled by PCR and in vitro transcription. Samples were analysed upon Affymetrix U133 Plus 2.0 Arrays. The expression of seven differentially expressed genes (CDH1, EMP1, DDR1, DVL1, KRT5, KRT6, KRT17) was verified by immunohistochemistry on tissue microarrays. Expression of ASPN mRNA was validated by in situ hybridization on frozen sections, and CTHRC1, ASPN and COL3A1 were tested by PCR.ResultsUsing GCOS pairwise comparison algorithm and rank products we have identified 84 named genes common to ILC versus normal cell types, 74 named genes common to IDC versus normal cell types, 78 named genes differentially expressed between normal ductal and lobular cells, and 28 named genes between IDC and ILC. Genes distinguishing between IDC and ILC are involved in epithelial-mesenchymal transition, TGF-beta and Wnt signaling. These changes were present in both tumor types but appeared to be more prominent in ILC. Immunohistochemistry for several novel markers (EMP1, DVL1, DDR1) distinguished large sets of IDC from ILC.ConclusionIDC and ILC can be differentiated both at the gene and protein levels. In this study we report two candidate genes, asporin (ASPN) and collagen triple helix repeat containing 1 (CTHRC1) which might be significant in breast carcinogenesis. Besides E-cadherin, the proteins validated on tissue microarrays (EMP1, DVL1, DDR1) may represent novel immunohistochemical markers helpful in distinguishing between IDC and ILC. Further studies with larger sets of patients are needed to verify the gene expression profiles of various histological types of breast cancer in order to determine molecular subclassifications, prognosis and the optimum treatment strategies.

Wnt Signaling Pathway in Mammary Gland Development and Carcinogenesis

The signaling pathway mediated by Wingless-type (Wnt) proteins is highly conserved in evolution. This pivotal pathway is known to regulate cell fate decisions, cell proliferation, morphology, migration, apoptosis, differentiation and stem cell self-renewal. It currently includes the canonical or Wnt/β-catenin pathway in which Wnt proteins bind to ‘frizzled’ receptors, which leads to downstream activation of gene transcription by β-catenin. Second, the noncanonical or β-catenin-independent pathways are now known to be mediated by three possible mechanisms: (1) the Wnt/Ca2+ pathway, (2) the Wnt/G protein signaling pathway, and (3) the Wnt/PCP or planar cell polarity pathway. Wnt signaling is implicated at several stages of mammary gland growth and differentiation, and possibly in the involution of mammary gland following lactation. Recent evidence suggests the role of Wnt signaling in human breast cancer involves elevated levels of nuclear and/or cytoplasmic β-catenin using immunohistochemistry, overexpression or downregulation of specific Wnt proteins, overexpression of CKII and sFRP4, downregulation of WIF-1 and sFRP1, as well as amplification of DVL-1. Further research is required to determine how Wnt signaling is involved in the development of different histological types of breast cancer and whether it promotes the viability of cancer stem cells or not.

Inhibition of the acetyltransferases p300 and CBP reveals a targetable function for p300 in the survival and invasion pathways of prostate cancer cell lines

Inhibitors of histone deacetylases have been approved for clinical application in cancer treatment. On the other hand, histone acetyltransferase (HAT) inhibitors have been less extensively investigated for their potential use in cancer therapy. In prostate cancer, the HATs and coactivators p300 and CBP are upregulated and may induce transcription of androgen receptor (AR)-responsive genes, even in the absence or presence of low levels of AR. To discover a potential anticancer effect of p300/CBP inhibition, we used two different approaches: (i) downregulation of p300 and CBP by specific short interfering RNA (siRNA) and (ii) chemical inhibition of the acetyltransferase activity by a newly developed small molecule, C646. Knockdown of p300 by specific siRNA, but surprisingly not of CBP, led to an increase of caspase-dependent apoptosis involving both extrinsic and intrinsic cell death pathways in androgen-dependent and castration-resistant prostate cancer cells. Induction of apoptosis was mediated by several pathways including inhibition of AR function and decrease of the nuclear factor kappa B (NF-κB) subunit p65. Furthermore, cell invasion was decreased upon p300, but not CBP, depletion and was accompanied by lower matrix metalloproteinase (MMP)-2 and MMP-9 transcriptions. Thus, p300 and CBP have differential roles in the processes of survival and invasion of prostate cancer cells. Induction of apoptosis in prostate cancer cells was confirmed by the use of C646. This was substantiated by a decrease of AR function and downregulation of p65 impairing several NF-κB target genes. Taken together, these results suggest that p300 inhibition may be a promising approach for the development of new anticancer therapies. Mol Cancer Ther; 10(9); 1644–55. ©2011 AACR.

Evaluation of candidate biomarkers to predict cancer cell sensitivity or resistance to PARP-1 inhibitor treatment

Impaired DNA damage response pathways may create vulnerabilities of cancer cells that can be exploited therapeutically. One such selective vulnerability is the sensitivity of BRCA1- or BRCA2-defective tumors (hence defective in DNA repair by homologous recombination, HR) to inhibitors of the poly(ADP-ribose) polymerase-1 (PARP-1), an enzyme critical for repair pathways alternative to HR. While promising, treatment with PARP-1 inhibitors (PARP-1i) faces some hurdles, including (1) acquired resistance, (2) search for other sensitizing, non-BRCA1/2 cancer defects and (3) lack of biomarkers to predict response to PARP-1i. Here we addressed these issues using PARP-1i on 20 human cell lines from carcinomas of the breast, prostate, colon, pancreas and ovary. Aberrations of the Mre11-Rad50-Nbs1 (MRN) complex sensitized cancer cells to PARP-1i, while p53 status was less predictive, even in response to PARP-1i combinations with camptothecin or ionizing radiation. Furthermore, monitoring PARsylation and Rad51 foci formation as surrogate markers for PARP activity and HR, respectively, supported their candidacy for biomarkers of PARP-1i responses. As to resistance mechanisms, we confirmed the role of the multidrug resistance efflux transporters and its reversibility. More importantly, we demonstrated that shRNA lentivirus-mediated depletion of 53BP1 in human BRCA1-mutant breast cancer cells increased their resistance to PARP-1i. Given the preferential loss of 53BP1 in BRCA-defective and triple-negative breast carcinomas, our findings warrant assessment of 53BP1 among candidate predictive biomarkers of response to PARPi. Overall, this study helps characterize genetic and functional determinants of cellular responses to PARP-1i and contributes to the search for biomarkers to exploit PARP inhibitors in cancer therapy.

The role of cancer-associated fibroblasts, solid stress and other microenvironmental factors in tumor progression and therapy resistance.

Tumors are not merely masses of neoplastic cells but complex tissues composed of cellular and noncellular elements. This review provides recent data on the main components of a dynamic system, such as carcinoma associated fibroblasts that change the extracellular matrix (ECM) topology, induce stemness and promote metastasis-initiating cells. Altered production and characteristics of collagen, hyaluronan and other ECM proteins induce increased matrix stiffness. Stiffness along with tumor growth-induced solid stress and increased interstitial fluid pressure contribute to tumor progression and therapy resistance. Second, the role of immune cells, cytokines and chemokines is outlined. We discuss other noncellular characteristics of the tumor microenvironment such as hypoxia and extracellular pH in relation to neoangiogenesis. Overall, full understanding of the events driving the interactions between tumor cells and their environment is of crucial importance in overcoming treatment resistance and improving patient outcome.

Urine markers in monitoring for prostate cancer.

The major advantages of urine-based assays are their noninvasive character and ability to monitor prostate cancer with heterogeneous foci. Almost all urine-detectable prostate-specific markers have been recently reviewed. For this reason, we focus here on only a few promising markers which have been independently evaluated (in particular PCA3, fusion genes, TERT, AMACR, GSTP1, MMP9 and VEGF) and very recent ones (ANXA3 and sarcosine). The emphasis is also on multiplex biomarker analysis and on microarray-based analysis of fusion genes. A combination of multiple urine biomarkers may be valuable in the case of men with persistently elevated serum prostate-specific antigen and a history of negative biopsies. The emerging urine tests should help in both early diagnosis of prostate cancer and identifying aggressive tumors for radical treatment.

Collagen triple helix repeat containing 1 protein, periostin and versican in primary and metastatic breast cancer: an immunohistochemical study

Background Collagen triple helix repeat containing 1 (CTHRC1) affects Wnt signalling, collagen deposition and bone formation. It is an extracellular matrix protein which is also abnormally expressed in the tumour microenvironment. CTHRC1 has not been studied in breast cancer by immunohistochemistry. Aims To examine expression of CTHRC1 together with periostin and versican in breast cancer patients and investigate its association with clinicopathological characteristics. Methods The formalin-fixed paraffin-embedded tissues of 173 invasive carcinomas (classified into WHO histotypes and luminal, triple negative and Her2 subtypes), as well as normal tissues, precursor lesions and metastatic lymph nodes were stained by relevant antibodies, assessed semiquantitatively by histoscore and statistically evaluated. Results Expression of CTHRC1, versican and periostin was significantly higher in breast cancer than in normal tissue or precursor lesions. CTHRC1 stromal expression was enhanced in triple negative cases and also in patients with bone metastasis. Periostin expression was high in primary tumours, in particular triple negative ones, and also in their lymph node metastases. Cox regression analysis showed that in patients with high periostin, the risk of bone metastases increased with increased CTHRC1 expression. Conclusions CTHRC1 and periostin play important roles in breast cancer progression. These preliminary results show that combined evaluation of CTHRC1 and periostin could serve as a potential marker for breast cancer bone metastasis; the other observations contribute to the description of the tumour microenvironment, with implications for lymph node and bone metastasis.

Transcriptional coactivators p300 and CBP stimulate estrogen receptor-beta signaling and regulate cellular events in prostate cancer†‡

Steroid receptor coactivators p300 and CBP are highly expressed in advanced prostate cancer. They potentiate activation of androgen receptor by androgens and anti‐androgens. In the present study, we have addressed the question whether these coactivators enhance activity of estrogen receptor‐beta (ER‐β), which is variably expressed in prostate cancers.

Telomere Attrition Occurs during Ex Vivo Expansion of Human Dental Pulp Stem Cells

We provide a detailed characteristic of stem cells isolated and expanded from the human dental pulp. Dental pulp stem cells express mesenchymal cell markers STRO-1, vimentin, CD29, CD44, CD73, CD90, CD166, and stem cell markers Sox2, nestin, and nucleostemin. They are multipotent as shown by their osteogenic and chondrogenic potential. We measured relative telomere length in 11 dental pulp stem cell lines at different passages by quantitative real-time PCR. Despite their large proliferative capacity, stable viability, phenotype, and genotype over prolonged cultivation, human dental pulp stem cells suffer from progressive telomere shortening over time they replicate in vitro. Relative telomere length (T/S) was inversely correlated with cumulative doubling time. Our findings indicate that excessive ex vivo expansion of adult stem cells should be reduced at minimum to avoid detrimental effects on telomere maintenance and measurement of telomere length should become a standard when certificating the status and replicative age of stem cells prior therapeutic applications.

A novel myoepithelial/progenitor cell marker in the breast?

Dear Editor, Neuroepithelial stem cell protein (Nestin) is an intermediate filament (IF) protein considered to be a marker of neural stem cells. Nestin expression has been confirmed in stem/ progenitor cells of the dermis, hair follicles, intestine, pancreas, bone marrow as well as in neural, muscle and other tissues (reviewed in [7]). Its expression is down regulated in the course of differentiation and subsequently replaced by another type of IF [3, 10]. Nestin expression has also been detected in the endothelium [11, 12], and we have noted the activation of nestin expression in newly formed human blood vessels [7]. The human nestin molecule has a homology with rodent nestin confirming its highly conserved nature (Fig. 2c) [5]. The myoepithelial cells of the breast form an outer layer of the terminal duct lobular unit. They play an active part in mammary gland branching morphogenesis [6], and their correct recognition and detection is crucial in the diagnosis of a number of pathological breast lesions [4, 8]. Several markers have been reported for the immunohistochemical detection of breast myoepithelial cells such as α-smooth muscle actin, smooth muscle myosin heavy chain, calponin, h-caldesmon, S100 protein, p63, CD10, maspin and specific cytokeratins 5, 7, 15 and 17 (reviewed in [2, 8]). However, the specificity and sensitivity of these markers vary widely. Of these, maspin [9] and p63 [1, 2, 13] are generally considered the most promising. In histopathological practice it is very often necessary to confirm a diagnosis using a wide battery of immunohistological stainings. Undoubtedly, the identification of new markers is desirable. We analysed the expression of nestin by an indirect immunohistochemical method (primary monoclonal antinestin antibody, clone 10C2, cat. no. MAB5326, Chemicon International, diluted 1:200; secondary staining system EnVision, Dako; antigen retrieval by microwave generator in citrate buffer pH 7.0; diaminobenzidine as chromogen) in 300 archival formalin-fixed and paraffin-embedded tissue samples of breast cancer and surrounding mammary tissue as well as in tissue microarrays consisting of 109 cases of breast cancer, developed for another project. Nestin expression of various intensities was seen in the cytoplasm of myoepithelial cells of almost all normal ducts and lobular acini found in the vicinity of tumours (Fig. 1a and b). The intensity of expression varied from strong/ marked (32 cases, ∼10%), to moderate/clearly visible (91 cases, ∼29%) to low/visible (113 cases, ∼38%) and to very low/visible in less than 50% cells only (64 cases, ∼21%). In our opinion, the reasons for the lower positive intensity in some cases can be explained by the fixation conditions. The specimens obtained by excision were generally more positive than specimens from the centre of the mastectomy. Pathological lesions like adenosis with atypical hyperplasia and in situ carcinoma exhibited very strong positivity (Fig. 1c and d), and the detection of nestin could also be used for the diagnosis of pseudo-invasion in some controversial cases (Fig. 1e). In invasive carcinomas, we found nestin positivity in a portion of epithelial cells in a minority (∼1/3) of cases. The positivity in the epithelial component was confirmed by simultaneous AE1/AE3 Virchows Arch (2007) 450:607–609 DOI 10.1007/s00428-007-0403-x