GLI1 confers profound phenotypic changes upon LNCaP prostate cancer cells that include the acquisition of a hormone independent state.

The GLI (GLI1/GLI2) transcription factors have been implicated in the development and progression of prostate cancer although our understanding of how they actually contribute to the biology of these common tumours is limited. We observed that GLI reporter activity was higher in normal (PNT-2) and tumourigenic (DU145 and PC-3) androgen-independent cells compared to androgen-dependent LNCaP prostate cancer cells and, accordingly, GLI mRNA levels were also elevated. Ectopic expression of GLI1 or the constitutively active ΔNGLI2 mutant induced a distinct cobblestone-like morphology in LNCaP cells that, regarding the former, correlated with increased GLI2 as well as expression of the basal/stem-like markers CD44, ß1-integrin, ΔNp63 and BMI1, and decreased expression of the luminal marker AR (androgen receptor). LNCaP-GLI1 cells were viable in the presence of the AR inhibitor bicalutamide and gene expression profiling revealed that the transcriptome of LNCaP-GLI1 cells was significantly closer to DU145 and PC-3 cells than to control LNCaP-pBP (empty vector) cells, as well as identifying LCN2/NGAL as a highly induced transcript which is associated with hormone independence in breast and prostate cancer. Functionally, LNCaP-GLI1 cells displayed greater clonal growth and were more invasive than control cells but they did not form colonies in soft agar or prostaspheres in suspension suggesting that they do not possess inherent stem cell properties. Moreover, targeted suppression of GLI1 or GLI2 with siRNA did not reverse the transformed phenotype of LNCaP-GLI1 cells nor did double GLI1/GLI2 knockdowns activate AR expression in DU145 or PC-3 cells. As such, early targeting of the GLI oncoproteins may hinder progression to a hormone independent state but a more detailed understanding of the mechanisms that maintain this phenotype is required to determine if their inhibition will enhance the efficacy of anti-hormonal therapy through the induction of a luminal phenotype and increased dependency upon AR function.

Alternative HER/PTEN/Akt pathway activation in HPV positive and negative penile carcinomas.

BACKGROUND: The pathogenesis of penile squamous cell carcinoma (PSCC) is not well understood, though risk factors include human papillomavirus (HPV). Disruption of HER/PTEN/Akt pathway is present in many cancers; however there is little information on its function in PSCC. We investigated HER family receptors and phosphatase and tension homolog (PTEN) in HPV-positive and negative PSCC and its impact on Akt activation using immunohistochemistry and fluorescent in situ hybridisation (FISH). METHODOLOGY/PRINCIPAL FINDINGS: 148 PSCCs were microarrayed and immunostained for phosphorylated EGFR (pEGFR), HER2, HER3, HER4, phosphorylated Akt (pAkt), Akt1 and PTEN proteins. EGFR and PTEN gene status were also evaluated using FISH. HPV presence was assessed by PCR. pEGFR expression was detected significantly less frequently in HPV-positive than HPV-negative tumours (p = 0.0143). Conversely, HER3 expression was significantly more common in HPV-positive cases (p = 0.0128). HER4, pAkt, Akt and PTEN protein expression were not related to HPV. HER3 (p = 0.0054) and HER4 (p = 0.0002) receptors significantly correlated with cytoplasmic Akt1 immunostaining. All three proteins positively correlated with tumour grade (HER3, p = 0.0029; HER4, p = 0.0118; Akt1, p = 0.0001). pEGFR expression correlated with pAkt but not with tumour grade or stage. There was no EGFR gene amplification. HER2 was not detected. PTEN protein expression was reduced or absent in 62% of tumours but PTEN gene copy loss was present only in 4% of PSCCs. CONCLUSIONS/SIGNIFICANCE: EGFR, HER3 and HER4 but not HER2 are associated with penile carcinogenesis. HPV-negative tumours tend to express significantly more pEGFR than HPV-positive cancers and this expression correlates with pAkt protein, indicating EGFR as an upstream regulator of Akt signalling in PSCC. Conversely, HER3 expression is significantly more common in HPV-positive cases and positively correlates with cytoplasmic Akt1 expression. HER4 and PTEN protein expression are not related to HPV infection. Our results suggest that PSCC patients could benefit from therapies developed to target HER receptors.

The retinoblastoma protein/p16 INK4A pathway but not p53 is disrupted by human papillomavirus in penile squamous cell carcinoma.

AIMS: The pathogenesis of penile squamous cell carcinoma (PSCC) is not well understood. Human papillomavirus (HPV) may be involved in carcinogenesis, but few studies have compared cell-cycle protein expression in HPV positive and negative cancers. The aim was to determine the extent of HPV infection in different histological subtypes of PSCC and its impact on the expression of key cell-cycle proteins: p53, p21, p16(INK4A) and retinoblastoma (RB) protein. METHODS AND RESULTS: One hundred and forty-eight PSCC samples were examined immunohistochemically for RB, p16(INK4A) , p53 and p21 protein expression. One hundred and two cases were typed for HPV by PCR. HPV DNA was detected in 56% of tumours, with HPV16 present in 81%. Basaloid tumours were related strongly to HPV infection (10 of 13), while verrucous were not (three of 13). Fifty-nine per cent (38 of 64) of usual type SCCs had HPV infection. RB protein correlated negatively (P<0.0001) and p16(INK4A) (P<0.0001) and p21 (P=0.0002) correlated positively with HPV infection. p53 did not correlate with HPV infection. CONCLUSIONS: HPV infection is present in more than half of penile cancers and it is responsible for RB pathway disruption. However, no link between HPV and p53 immunodetection was found. Only basaloid and half of usual-type PSSCs correlate with HPV infection, confirming possible separate aetiologies for those tumours.

HPV infection and immunochemical detection of cell-cycle markers in verrucous carcinoma of the penis.

Penile verrucous carcinoma is a rare disease and little is known of its aetiology or pathogenesis. In this study we examined cell-cycle proteins expression and correlation with human papillomavirus infection in a series of 15 pure penile verrucous carcinomas from a single centre. Of 148 penile tumours, 15 (10%) were diagnosed as pure verrucous carcinomas. The expression of the cell-cycle-associated proteins p53, p21, RB, p16(INK4A) and Ki67 were examined by immunohistochemistry. Human papillomavirus infection was determined by polymerase chain reaction to identify a wide range of virus types. The expression of p16(INK4A) and Ki67 was significantly lower in verrucous carcinoma than in usual type squamous cell carcinoma, whereas the expression of p53, p21 and RB was not significantly different. p53 showed basal expression in contrast to usual type squamous cell carcinoma. Human papillomavirus infection was present in only 3 out of 13 verrucous carcinomas. Unique low-risk, high-risk and mixed viral infections were observed in each of the three cases. In conclusion, lower levels of p16(INK4A) and Ki67 expressions differentiate penile verrucous carcinoma from usual type squamous cell carcinoma. The low Ki67 index reflects the slow-growing nature of verrucous tumours. The low level of p16(INK4A) expression and human papillomavirus detection suggests that penile verrucous carcinoma pathogenesis is unrelated to human papillomavirus infection and the oncogenes and tumour suppressor genes classically altered by virus infection.

WNT signaling regulates self-renewal and differentiation of prostate cancer cells with stem cell characteristics.

Prostate cancer cells with stem cell characteristics were identified in human prostate cancer cell lines by their ability to form from single cells self-renewing prostaspheres in non-adherent cultures. Prostaspheres exhibited heterogeneous expression of proliferation, differentiation and stem cell-associated makers CD44, ABCG2 and CD133. Treatment with WNT inhibitors reduced both prostasphere size and self-renewal. In contrast, addition of Wnt3a caused increased prostasphere size and self-renewal, which was associated with a significant increase in nuclear beta-catenin, keratin 18, CD133 and CD44 expression. As a high proportion of LNCaP and C4-2B cancer cells express androgen receptor we determined the effect of the androgen receptor antagonist bicalutamide. Androgen receptor inhibition reduced prostasphere size and expression of PSA, but did not inhibit prostasphere formation. These effects are consistent with the androgen-independent self-renewal of cells with stem cell characteristics and the androgen-dependent proliferation of transit amplifying cells. As the canonical WNT signaling effector beta-catenin can also associate with the androgen receptor, we propose a model for tumour propagation involving a balance between WNT and androgen receptor activity. That would affect the self-renewal of a cancer cell with stem cell characteristics and drive transit amplifying cell proliferation and differentiation. In conclusion, we provide evidence that WNT activity regulates the self-renewal of prostate cancer cells with stem cell characteristics independently of androgen receptor activity. Inhibition of WNT signaling therefore has the potential to reduce the self-renewal of prostate cancer cells with stem cell characteristics and improve the therapeutic outcome.

Human papillomavirus is detected in transitional cell carcinoma arising in renal transplant recipients.

AIMS: We investigated the role of human papillomavirus (HPV) in the development of transitional cell carcinoma (TCC) arising in renal transplant recipients. METHODS: Genomic DNA was extracted from 10 microm paraffin embedded sections of five TCCs arising in five renal transplant recipients using the QIAamp DNA mini kit according to the manufacturer's instructions. beta-globin PCR was performed to test DNA adequacy. Samples were tested for the presence of HPV DNA by broad spectrum HPV PCR method using non-biotinylated SPF10 primers (SPF1A, SPF1B, SPF1C, SPF1D, SPF2B, SPF2D) which amplify a short 65 bp fragment. Positive bands were identified on a 3% gel. Positive samples underwent a second HPV PCR and were amplified using biotinylated SPF10 primer set, which amplifies the same 65 bp region of the L1 open reading frame. INNO-LiPA line probe assay was then performed to genotype the samples which uses a reverse hybridisation principle. RESULTS: Four of five TCCs examined were positive for HPV. The high risk HPV16 was detected in three cases whereas in the fourth case an unclassifiable HPV genotype was present. In all DNA samples, beta-globin amplification was successful. CONCLUSIONS: Our results indicate that HPV and in particular HPV16 may play an aetiological role in the development of TCC in renal transplant patients.

Detection of TMPRSS2:ERG fusion gene in circulating prostate cancer cells.

AIM: To investigate the existence of TMPRSS2:ERG fusion gene in circulating tumor cells (CTC) from prostate cancer patients and its potential in monitoring tumor metastasis. METHODS: We analyzed the frequency of TMPRSS2:ERG and TMPRSS2:ETV1 transcripts in 27 prostate cancer biopsies from prostatectomies, and TMPRSS2:ERG transcripts in CTC isolated from 15 patients with advanced androgen independent disease using reverse transcription polymerase chain reaction (RT-PCR). Fluorescence in situ hybridization (FISH) was applied to analyze the genomic truncation of ERG, which is the result of TMPRSS2:ERG fusion in 10 of the 15 CTC samples. RESULTS: TMPRSS2:ERG transcripts were found in 44% of our samples, but we did not detect expression of TMPRSS2:ETV1. Using FISH analysis we detected chromosomal rearrangements affecting the ERG gene in 6 of 10 CTC samples, including 1 case with associated TMPRSS2:ERG fusion at the primary site. However, TMPRSS2:ERG transcripts were not detected in any of the 15 CTC samples, including the 10 cases analyzed by FISH. CONCLUSION: Although further study is required to address the association between TMPRSS2:ERG fusion and prostate cancer metastasis, detection of genomic truncation of the ERG gene by FISH analysis could be useful for monitoring the appearance of CTC and the potential for prostate cancer metastasis.

Inhibition of androgen-independent prostate cancer cell growth is enhanced by combination therapy targeting Hedgehog and ErbB signalling.

BACKGROUND: Prostate cancer is a leading cause of male cancer specific mortality. When cure by radical prostatectomy is not possible the next line of prostate cancer treatment is androgen deprivation. However prolonged androgen deprivation often results in relapse and androgen-independent prostate cancer that is inevitably fatal despite optimal chemotherapy. The Hedgehog signalling pathway has recently been implicated in prostate cancer development and metastasis. EGFR or ErbB2 expression has been also correlated with androgen independence, shorter survival and metastasis. RESULTS: We determined that the Hedgehog and ErbB signalling pathways are active in circulating tumour cells isolated from androgen-independent prostate cancer patients and in the androgen-independent prostate cancer cell line LNCaP C4-2B. As a basis for synergistic chemotherapy protocols combinations of the Hedgehog specific inhibitor cyclopamine and the ErbB signalling inhibitors gefitinib or lapatinib were tested in this study. Androgen-independent prostate cancer cell growth was inhibited by a SMO inhibitor (cyclopamine) which blocks Hedgehog signalling and by ErbB inhibitors (gefitinib and lapatinib). The isobologram and combination index method of Chou and Talalay was used to evaluate drug interactions. Synergistic antiproliferation effects were observed when the Hedgehog and ErbB inhibitors were combined. CONCLUSION: Androgen-independent prostate cancer cell proliferation was associated with activity of the Hedgehog and ErbB signalling pathways. Cyclopamine, gefitinib or lapatinib treatment significantly decreased the proliferation of androgen-independent prostate cancer cells. The Hedgehog pathway therefore represents a promising new therapeutic target in androgen-independent prostate cancer. Synergistic effects were observed when Hedgehog and ErbB inhibitors were used together. This study may have clinical implications for improving the treatment of advanced prostate cancer.

Hedgehog signalling in androgen independent prostate cancer.

OBJECTIVES: Androgen-deprivation therapy effectively shrinks hormone-naïve prostate cancer, both in the prostate and at sites of distant metastasis. However prolonged androgen deprivation generally results in relapse and androgen-independent tumour growth, which is inevitably fatal. The molecular events that enable prostate cancer cells to proliferate in reduced androgen conditions are poorly understood. Here we investigate the role of Hedgehog signalling in androgen-independent prostate cancer (AIPC). METHODS: Activity of the Hedgehog signalling pathway was analysed in cultured prostate cancer cells, and circulating prostate tumour cells were isolated from blood samples of patients with AIPC. RESULTS: AIPC cells were derived through prolonged culture in reduced androgen conditions, modelling hormone therapy in patients, and expressed increased levels of Hedgehog signalling proteins. Exposure of cultured AIPC cells to cyclopamine, which inhibits Hedgehog signalling, resulted in inhibition of cancer cell growth. The expression of the Hedgehog receptor PTCH and the highly prostate cancer-specific gene DD3(PCA3) was significantly higher in circulating prostate cancer cells isolated from patients with AIPC compared with samples prepared from normal individuals. There was an association between PTCH and DD3(PCA3) expression and the length of androgen-ablation therapy. CONCLUSIONS: Our data are consistent with reports implicating overactivity of Hedgehog signalling in prostate cancer and suggest that Hedgehog signalling contributes to the androgen-independent growth of prostate cancer cells. As systemic anti-Hedgehog medicines are developed, the Hedgehog pathway will become a potential new therapeutic target in advanced prostate cancer.

International study into the use of intermittent hormone therapy in the treatment of carcinoma of the prostate: a meta-analysis of 1446 patients.

OBJECTIVE: To review pooled phase II data to identify features of different regimens of intermittent hormone therapy (IHT), developed to reduce the morbidity of treating metastatic prostate cancer, and which carries a theoretical advantage of delaying the onset of androgen-independent prostate cancer, (AIPC) that are associated with success, highlighting features which require exploration with prospective trials to establish the best strategies for using this treatment. METHODS: Individual data were collated on 1446 patients with adequate information, from 10 phase II studies with >50 cases, identified through Pubmed. RESULTS: Univariate and multivariate Cox proportional hazard models were developed to predict treatment success with a high degree of statistical success. The prostate-specific antigen (PSA) nadir, the PSA threshold to restart treatment, and medication type and duration, were important predictors of outcome. CONCLUSIONS: The duration of biochemical remission after a period of HT is a durable early indicator of how rapidly AIPC and death will occur, and will make a useful endpoint in future trials to investigate the best ways to use IHT based on the important treatment cycling variables described above. Patients spent a mean of 39% of the time off treatment. The initial PSA level and PSA nadir allow the identification of patients with prostate cancer in whom it might be possible to avoid radical therapy.

Distinct epidermal stem cell compartments are maintained by independent niche microenvironments.

The mammalian epidermis is a stratified, multilayered epithelium, consisting of the interfollicular epidermis and associated appendages, which extend into the dermis and include hair follicles, sebaceous glands, and sweat glands. Stem cells are essential for the maintenance of this tissue and are also potential sources of multipotent adult precursor cells. Stem cell populations occupying specific locations or niches have been identified in the interfollicular epidermis, the hair follicle and the sebaceous gland. Recent research has focused on how the stem cell niches provide specific sites where stem cells can reside indefinitely and undergo self-renewal or differentiation into specific cell lineages, as required for epidermal replenishment or hair follicle growth.

Beta-catenin and Hedgehog signal strength can specify number and location of hair follicles in adult epidermis without recruitment of bulge stem cells.

Using K14deltaNbeta-cateninER transgenic mice, we show that short-term, low-level beta-catenin activation stimulates de novo hair follicle formation from sebaceous glands and interfollicular epidermis, while only sustained, high-level activation induces new follicles from preexisting follicles. The Hedgehog pathway is upregulated by beta-catenin activation, and inhibition of Hedgehog signaling converts the low beta-catenin phenotype to wild-type epidermis and the high phenotype to low. beta-catenin-induced follicles contain clonogenic keratinocytes that express bulge markers; the follicles induce dermal papillae and provide a niche for melanocytes, and they undergo 4OHT-dependent cycles of growth and regression. New follicles induced in interfollicular epidermis are derived from that cellular compartment and not through bulge stem cell migration or division. These results demonstrate the remarkable capacity of adult epidermis to be reprogrammed by titrating beta-catenin and Hedgehog signal strength and establish that cells from interfollicular epidermis can acquire certain characteristics of bulge stem cells.

Transient activation of FOXN1 in keratinocytes induces a transcriptional programme that promotes terminal differentiation: contrasting roles of FOXN1 and Akt.

The forkhead transcription factor FOXN1 is required for normal cutaneous and thymic epithelial development. Mutations in FOXN1 give rise to the nude phenotype in mice, rats and man. However, the genes that are regulated by FOXN1 are unknown. To investigate FOXN1 function we expressed an inducible form of the protein, FOXN1ER, that is activated by 4-hydroxytamoxifen in primary human epidermal keratinocytes. Transient activation of FOXN1 decreased the proportion of keratinocytes that formed actively growing clones attributable to stem cell founders and increased the number of abortive clones, without inducing apoptosis. Within 24 hours the majority of cells had initiated terminal differentiation, as assessed by involucrin expression. We performed a cDNA microarray experiment to analyse changes in the transcription of approximately 6,000 genes. Following FOXN1 activation we detected increases of two fold or greater in the RNA levels of over 30 genes. Genes promoting growth arrest, survival and differentiation featured prominently and markers of early events in keratinocyte differentiation were also detected. Since one of the induced genes was Akt we investigated whether Akt played a role in terminal differentiation. Activation of PI 3-kinase but not Akt was necessary for FOXN1-induced differentiation. In reconstituted epidermis FOXN1 promoted early stages of terminal differentiation whereas Akt activation was sufficient to induce late stages, including formation of the cornified layers. These results establish a role for FOXN1 in initiation of terminal differentiation and implicate Akt in subsequent events.

Transient activation of beta-catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles but continuous activation is required to maintain hair follicle tumours.

When beta-catenin signalling is disturbed from mid-gestation onwards lineage commitment is profoundly altered in postnatal mouse epidermis. We have investigated whether adult epidermis has the capacity for beta-catenin-induced lineage conversion without prior embryonic priming. We fused N-terminally truncated, stabilised beta-catenin to the ligand-binding domain of a mutant oestrogen receptor (DeltaNbeta-cateninER). DeltaNbeta-cateninER was expressed in the epidermis of transgenic mice under the control of the keratin 14 promoter and beta-catenin activity was induced in adult epidermis by topical application of 4-hydroxytamoxifen (4OHT). Within 7 days of daily 4OHT treatment resting hair follicles were recruited into the hair growth cycle and epithelial outgrowths formed from existing hair follicles and from interfollicular epidermis. The outgrowths expressed Sonic hedgehog, Patched and markers of hair follicle differentiation, indicative of de novo follicle formation. The interfollicular epidermal differentiation program was largely unaffected but after an initial wave of sebaceous gland duplication sebocyte differentiation was inhibited. A single application of 4OHT was as effective as repeated doses in inducing new follicles and growth of existing follicles. Treatment of epidermis with 4OHT for 21 days resulted in conversion of hair follicles to benign tumours resembling trichofolliculomas. The tumours were dependent on continuous activation of beta-catenin and by 28 days after removal of the drug they had largely regressed. We conclude that interfollicular epidermis and sebaceous glands retain the ability to be reprogrammed in adult life and that continuous beta-catenin signalling is required to maintain hair follicle tumours.

Pak1 phosphorylation on t212 affects microtubules in cells undergoing mitosis.

The Pak kinases are targets of the Rho GTPases Rac and Cdc42, which regulate cell shape and motility. It is increasingly apparent that part of this function is due to the effect Pak kinases have on microtubule organization and dynamics. Recently, overexpression of Xenopus Pak5 was shown to enhance microtubule stabilization, and it was shown that mammalian Pak1 may inhibit a microtubule-destabilizing protein, Op18/Stathmin. We have identified a specific phosphorylation site on mammalian Pak1, T212, which is targeted by the neuronal p35/Cdk5 kinase. Pak1 phosphorylated on T212, Pak1T212(PO(4)), is enriched in axonal growth cones and colocalizes with small peripheral bundles of microtubules. Cortical neurons overexpressing a Pak1A212 mutant display a tangled neurite morphology, which suggests that the microtubule cytoskeleton is affected. Here, we show that cyclin B1/Cdc2 phosphorylates Pak1 in cells undergoing mitosis. In the developing cortex and in cultured fibroblasts, Pak1T212(PO(4)) is enriched in microtubule-organizing centers and along parts of the spindles. In living cells, a peptide mimicking phosphorylated T212 accumulates at the centrosomes and spindles and causes an increased length of astral microtubules during metaphase or following nocodazole washout. Together these results suggest that similar signaling pathways regulate microtubule dynamics in a remodeling axonal growth cone and during cell division.