BackgroundEvery year approximately 74,000 women die of endometrial cancer, mainly due to recurrent or metastatic disease. The presence of tumor infiltrating lymphocytes (TILs) as well as progesterone receptor (PR) positivity has been correlated with improved prognosis. This study describes two mechanisms by which progesterone inhibits metastatic spread of endometrial cancer: by stimulating T-cell infiltration and by inhibiting epithelial-to-mesenchymal cell transition (EMT).Methodology and Principal FindingsParaffin sections from patients with (n = 9) or without (n = 9) progressive endometrial cancer (recurrent or metastatic disease) were assessed for the presence of CD4+ (helper), CD8+ (cytotoxic) and Foxp3+ (regulatory) T-lymphocytes and PR expression. Progressive disease was observed to be associated with significant loss of TILs and loss of PR expression. Frozen tumor samples, used for genome-wide expression analysis, showed significant regulation of pathways involved in immunesurveillance, EMT and metastasis. For a number of genes, such as CXCL14, DKK1, DKK4, PEG10 and WIF1, quantitive RT-PCR was performed to verify up- or downregulation in progressive disease. To corroborate the role of progesterone in regulating invasion, Ishikawa(IK) endometrial cancer cell lines stably transfected with PRA (IKPRA), PRB(IKPRB) and PRA+PRB (IKPRAB) were cultured in presence/absence of progesterone (MPA) and used for genome-wide expression analysis, Boyden- and wound healing migration assays, and IHC for known EMT markers. IKPRB and IKPRAB cell lines showed MPA induced inhibition of migration and loss of the mesenchymal marker vimentin at the invasive front of the wound healing assay. Furthermore, pathway analysis of significantly MPA regulated genes showed significant down regulation of important pathways involved in EMT, immunesuppression and metastasis: such as IL6-, TGF-β and Wnt/β-catenin signaling.ConclusionIntact progesterone signaling in non-progressive endometrial cancer seems to be an important factor stimulating immunosurveilance and inhibiting transition from an epithelial to a more mesenchymal, more invasive phenotype.

To study the functional differences between the two progesterone receptor isoforms (hPRA and hPRB) in human endometrial cancer, two new endometrial carcinoma cell lines were created-one expressing hPRA and one expressing hPRB.A well-differentiated, hPR-negative Ishikawa cell line was stably transfected with either hPRA or hPRB cDNA. Transfected cells were selected, and two cell lines expressing approximately equal amounts of receptor were isolated-one expressing hPRA (PRA-14) and one expressing hPRB (PRB-59). Cell growth experiments revealed a growth-inhibitory response to progestins (MPA and R5020) in the PRB-59 cells but not in the PRA-14 cells. Differences in expression of genes targeted by the two isoforms were studied using a cDNA expression array technique. A different set of genes appeared to be progesterone regulated in the PRA-14 cells than in the PRB-59 cells. None of the genes were regulated by both hPRA and hPRB. Insulin-like growth factor binding protein 3 expression was studied in more detail as an example of a gene regulated in PRB-59 cells but not in PRA-14 cells. We established a new model to study functional differences between the two hPR isoforms in human endometrial carcinoma cells. This model revealed distinctive differences in target gene regulation between the two hPR isoforms. Moreover, antiproliferative actions of progesterone on human endometrial cancer cells could be observed only in the PRB-expressing cell line.

In advanced prostate cancer, cellular changes occur leading to a transition from androgen-dependent to androgen-independent growth. During this transition, proliferation of androgendependent prostate cancer cells becomes more and more dependent on growth factors, like the epidermal growth factor (EGF). Endocytosis of growth factor receptors, one of the mechanisms that controls growth factor signalling, was observed to be markedly changed in advanced metastatic prostate cancer. Internalisation and signalling of EGF receptors was examined in different prostate cancer cell lines, in relation to the expression level of the endocytosis-related REPS2 gene. It was observed that a high level of REPS2 correlates with reduced EGF-internalisation. To investigate this more thoroughly, prostate cancer cells with inducible REPS2 expression were generated. Using these cells, it was found that REPS2-induction indeed results in reduction of EGF-internalisation. Furthermore, when EGF receptor signalling was evaluated, by examination of mRNA expression for several EGF-responsive genes (EGF receptor, EGR-1, Fos and Jun), it was observed that induced expression of REPS2 exerts an inhibiting effect on this signalling. From these experiments, it is concluded that increased REPS2 expression negatively affects EGF receptor internalisation and subsequent signalling. Therefore, decreased REPS2 expression during prostate cancer progression, observed in earlier work, may result in enhanced EGF receptor expression and signalling, which could add to the androgen-independent state of advanced prostate cancer. Key words: REPS2; prostate cancer; endocytosis; EGF signalling Treatment of prostate cancer involves surgery of the affected region of the gland and treatment of advanced prostate cancer focuses on inhibition of growth inducing signals, mainly by androgen ablation. However, given enough time, the tumour will switch from androgens to growth factors as primary regulators of proliferation. This transition leads to therapy-resistance of the cancer cells and eventually will result in recurrence of the disease. 1 Epidermal growth factor (EGF) and transforming growth factor alpha are potent mitogens involved in autocrine regulation of proliferation, angiogenesis and metastatic spread of advanced prostate cancer. 2 The EGF receptor can bind many ligands, among which EGF and TGF-␣. 3 Upon ligand-binding, the receptor dimerizes and is activated by autophosphorylation of tyrosine residues in the intracellular tail of the receptor. Subsequent activation of well-characterised signal transduction pathways (MAPK) results in proliferation and survival of tumour cells. 4,5 During prostate cancer progression, the EGF receptor expression level is regulated by transcriptional and posttranscriptional mechanisms, including upregulation of receptor mRNA and downregulation of EGF receptor protein by EGF. Intracellular trafficking of the EGF receptor was suggested to cause the overall degradation of the EGF receptor protein in prostate cancer cells. 6 This traffi...

Tamoxifen treatment for breast cancer increases proliferation of the endometrium, resulting in an enhanced prevalence of endometrial pathologies, including endometrial cancer. An exploratory study was performed to begin to understand the molecular mechanism of tamoxifen action in the endometrium. Gene-expression profiles were generated of endometrial samples of tamoxifen users and compared with matched controls. The pathological classification of samples from both groups included atrophic/inactive endometrium and endometrial polyps. Unsupervised clustering revealed that samples of tamoxifen users were, irrespective of pathological classification, fairly similar and consequently form a subgroup distinct from the matched controls. Using SAM analysis (a statistical method to select genes differentially expressed between groups), 256 differentially expressed genes were selected between the tamoxifen and control groups. Upon comparing these genes with oestrogen-regulated genes, identified under similar circumstances, 95% of the differentially expressed genes turned out to be tamoxifen-specific. Finally, construction of a gene-expression network of the differentially expressed genes revealed that 69 genes centred around five well-known genes: TP53, RELA, MYC, epidermal growth factor receptor and b-catenin. This could indicate that these well-known genes, and the pathways in which they function, are important for tamoxifencontrolled proliferation of the endometrium.

Regulation of amphiregulin coincides with the described in vivo effect of the four ligands on the endometrium. Therefore, it is possible that modulation of EGF receptor signaling is a significant player in estrogen-agonistic growth of the endometrium and needs to be investigated further.

Reintroduction of PRs into Ishikawa cells rescued progesterone responsiveness in these cells. Furthermore, using these human endometrial cancer subcell lines, clear and distinct functional differences between the PR isoforms were observed.

To study the functional differences between the two progesterone receptor isoforms (hPRA and hPRB) in human endometrial cancer, two new endometrial carcinoma cell lines were created-one expressing hPRA and one expressing hPRB.A well-differentiated, hPR-negative Ishikawa cell line was stably transfected with either hPRA or hPRB cDNA. Transfected cells were selected, and two cell lines expressing approximately equal amounts of receptor were isolated-one expressing hPRA (PRA-14) and one expressing hPRB (PRB-59). Cell growth experiments revealed a growth-inhibitory response to progestins (MPA and R5020) in the PRB-59 cells but not in the PRA-14 cells. Differences in expression of genes targeted by the two isoforms were studied using a cDNA expression array technique. A different set of genes appeared to be progesterone regulated in the PRA-14 cells than in the PRB-59 cells. None of the genes were regulated by both hPRA and hPRB. Insulin-like growth factor binding protein 3 expression was studied in more detail as an example of a gene regulated in PRB-59 cells but not in PRA-14 cells. We established a new model to study functional differences between the two hPR isoforms in human endometrial carcinoma cells. This model revealed distinctive differences in target gene regulation between the two hPR isoforms. Moreover, antiproliferative actions of progesterone on human endometrial cancer cells could be observed only in the PRB-expressing cell line.

Usual type VIN is a premalignant disorder caused by persistent HPV infection. High prevalence of VIN in immuno-suppressed women suggests that a good innate and adaptive immune response is important for defense against HPV. Here, we explored expression levels of chemokines and related these to the presence or absence of immuno-competent cells (dendritic and T-cells) in affected (HPV-positive VIN) and non-affected (HPV-negative) vulvar tissues from the same patients. Combining microarray data with quantitative real-time RT-PCR, it was observed that several important chemokines were differentially expressed between VIN and control samples (up-regulation of IL8, CXCL10, CCL20 and CCL22 and down-regulation of CXCL12, CCL21 and CCL14). Furthermore, an increased number of mature dendritic cells (CD208 1 ) seemed to be bottled up in the dermis, and although a T-cell response (increased CD4 1 and CD8 1 cells) was observed in VIN, a much larger response is required to clear the infection. In summary, it seems that most mature dendritic cells do not receive the proper chemokine signal for migration and will stay in the dermis, not able to present viral antigen to naive T-cells in the lymph node. Consequently the adaptive immune response diminishes, resulting in a persistent HPV infection with increased risk for neoplasia. ' 2008 Wiley-Liss, Inc.Key words: vulvar intraepithelial neoplasia; HPV; immune response; chemokines During the last decade, vulvar intraepithelial neoplasia (VIN), a premalignant disorder, has been diagnosed with increasing frequency in relatively young women in western countries. 1 This rise in incidence is presumably due to the rise in human papilloma virus (HPV) infections. Recently, more than 100 types of HPV are identified and the life-time risk for infection with one of these types is around 80%. 2,3 The different types of HPV are subdivided into low-risk (non-oncogenic, e.g., HPV 6 and 11) and high-risk HPV (oncogenic, e.g., HPV 16, 18 and 33). Around 40% of young, sexually active women are infected with the latter, a high-risk HPV. 4 Fortunately, most women are able to clear this infection, and less than 10% of infected women develop a persistent HPV infection, which is one of the leading causes of preneoplastic and neoplastic lesions in the female genital tract, including VIN. 5 The host immune response is of critical importance in determining the progression or regression of HPV-related VIN, because being immuno-compromised is a risk factor. For example, VIN is observed more often in women infected with HIV, and in women who are on systemic steroids to treat autoimmune diseases or to prevent rejection after transplantation. 6,7 Smoking is also a risk factor, because it results in a decreased local immune response in the epithelium by reducing the number of intraepithelial Langerhans cells. 8,9 After infection, the host develops a virus-specific cell-mediated immune response, which, in many instances, will lead to clearance of the virus within 1 year. 10 This immunological defense system consis...