Identification of the human prostatic carcinoma oncogene PTI-1 by rapid expression cloning and differential RNA display.

Elucidating the relevant genomic changes mediating development and evolution of prostate cancer is paramount for effective diagnosis and therapy. A putative dominant-acting nude mouse prostatic carcinoma tumor-inducing gene, PTI-1, has been cloned that is expressed in patient-derived human prostatic carcinomas but not in benign prostatic hypertrophy or normal prostate tissue. PTI-1 was detected by cotransfecting human prostate carcinoma DNA into CREF-Trans 6 cells, inducing tumors in nude mice, and isolating genes displaying increased expression in tumor-derived cells by using differential RNA display (DD). Screening a human prostatic carcinoma (LNCaP) cDNA library with a 214-bp DNA fragment found by DD permitted the cloning of a full-length 2.0-kb PTI-1 cDNA. Sequence analysis indicates that PTI-1 is a gene containing a 630-bp 5' sequence and a 3' sequence homologous to a truncated and mutated form of human elongation factor 1 alpha. In vitro translation demonstrates that the PTI-1 cDNA encodes a predominant approximately 46-kDa protein. Probing Northern blots with a DNA fragment corresponding to the 5' region of PTI-1 identifies multiple PTI-1 transcripts in RNAs from human carcinoma cell lines derived from the prostate, lung, breast, and colon. In contrast, PTI-1 RNA is not detected in human melanoma, neuroblastoma, osteosarcoma, normal cerebellum, or glioblastoma multiforme cell lines. By using a pair of primers recognizing a 280-bp region within the 630-bp 5' PTI-1 sequence, reverse transcription-PCR detects PTI-1 expression in patient-derived prostate carcinomas but not in normal prostate or benign hypertrophic prostate tissue. In contrast, reverse transcription-PCR detects prostate-specific antigen expression in all of the prostate tissues. These results indicate that PTI-1 may be a member of a class of oncogenes that could affect protein translation and contribute to carcinoma development in human prostate and other tissues. The approaches used, rapid expression cloning with the CREF-Trans 6 system and the DD strategy, should prove widely applicable for identifying and cloning additional human oncogenes.

Alterazione della proteostasi a livello del reticolo endoplasmatico: un potenziale target terapeutico nel carcinoma prostatico

Nel sesso maschile il carcinoma della prostata (CaP) è la neoplasia più frequente ed è tra le prime cause di morte per tumore. Ad oggi, sono disponibili diverse strategie terapeutiche per il trattamento del CaP, ma, come comprovato dall’ancora alta mortalità, spesso queste sono inefficaci, a causa soprattutto dello sviluppo di fenomeni di resistenza da parte delle cellule tumorali. La ricerca si sta quindi focalizzando sulla caratterizzazione di tali meccanismi di resistenza e, allo stesso tempo, sull’individuazione di combinazioni terapeutiche che siano più efficaci e capaci di superare queste resistenze. Le cellule tumorali sono fortemente dipendenti dai meccanismi connessi con l’omeostasi proteica (proteostasi), in quanto sono sottoposte a numerosi stress ambientali (ipossia, carenza di nutrienti, esposizione a chemioterapici, ecc.) e ad un’aumentata attività trascrizionale, entrambi fattori che causano un accumulo intracellulare di proteine anomale e/o mal ripiegate, le quali possono risultare dannose per la cellula e vanno quindi riparate o eliminate efficientemente. La cellula ha sviluppato diversi sistemi di controllo di qualità delle proteine, tra cui gli chaperon molecolari, il sistema di degradazione associato al reticolo endoplasmatico (ERAD), il sistema di risposta alle proteine non ripiegate (UPR) e i sistemi di degradazione come il proteasoma e l’autofagia. Uno dei possibili bersagli in cellule tumorali secretorie, come quelle del CaP, è rappresentato dal reticolo endoplasmatico (RE), organello intracellulare deputato alla sintesi, al ripiegamento e alle modificazioni post-traduzionali delle proteine di membrana e secrete. Alterazioni della protestasi a livello del RE inducono l’UPR, che svolge una duplice funzione nella cellula: primariamente funge da meccanismo omeostatico e di sopravvivenza, ma, quando l’omeostasi non è più ripristinabile e lo stimolo di attivazione dell’UPR cronicizza, può attivare vie di segnalazione che conducono alla morte cellulare programmata. La bivalenza, tipica dell’UPR, lo rende un bersaglio particolarmente interessante per promuovere la morte delle cellule tumorali: si può, infatti, sfruttare da una parte l’inibizione di componenti dell’UPR per abrogare i meccanismi adattativi e di sopravvivenza e dall’altra si può favorire il sovraccarico dell’UPR con conseguente induzione della via pro-apoptotica. Le catechine del tè verde sono composti polifenolici estratti dalle foglie di Camellia sinesis che possiedono comprovati effetti antitumorali: inibiscono la proliferazione, inducono la morte di cellule neoplastiche e riducono l’angiogenesi, l’invasione e la metastatizzazione di diversi tipi tumorali, tra cui il CaP. Diversi studi hanno osservato come il RE sia uno dei bersagli molecolari delle catechine del tè verde. In particolare, recenti studi del nostro gruppo di ricerca hanno messo in evidenza come il Polyphenon E (estratto standardizzato di catechine del tè verde) sia in grado, in modelli animali di CaP, di causare un’alterazione strutturale del RE e del Golgi, un deficit del processamento delle proteine secretorie e la conseguente induzione di uno stato di stress del RE, il quale causa a sua volta l’attivazione delle vie di segnalazione dell’UPR. Nel presente studio su due diverse linee cellulari di CaP (LNCaP e DU145) e in un nostro precedente studio su altre due linee cellulari (PNT1a e PC3) è stato confermato che il Polyphenon E è capace di indurre lo stress del RE e di determinare l’attivazione delle vie di segnalazione dell’UPR, le quali possono fungere da meccanismo di sopravvivenza, ma anche contribuire a favorire la morte cellulare indotta dalle catechine del tè verde (come nel caso delle PC3). Considerati questi effetti delle catechine del tè verde in qualità di induttori dell’UPR, abbiamo ipotizzato che la combinazione di questi polifenoli bioattivi e degli inibitori del proteasoma, anch’essi noti attivatori dell’UPR, potesse comportare un aggravamento dell’UPR stesso tale da innescare meccanismi molecolari di morte cellulare programmata. Abbiamo quindi studiato l’effetto di tale combinazione in cellule PC3 trattate con epigallocatechina-3-gallato (EGCG, la principale tra le catechine del tè verde) e due diversi inibitori del proteasoma, il bortezomib (BZM) e l’MG132. I risultati hanno dimostrato, diversamente da quanto ipotizzato, che l’EGCG quando associato agli inibitori del proteasoma non produce effetti sinergici, ma che anzi, quando viene addizionato al BZM, causa una risposta simil-antagonistica: si osserva infatti una riduzione della citotossicità e dell’effetto inibitorio sul proteasoma (accumulo di proteine poliubiquitinate) indotti dal BZM, inoltre anche l’induzione dell’UPR (aumento di GRP78, p-eIF2α, CHOP) risulta ridotta nelle cellule trattate con la combinazione di EGCG e BZM rispetto alle cellule trattate col solo BZM. Gli stessi effetti non si osservano invece nelle cellule PC3 trattate con l’EGCG in associazione con l’MG132, dove non si registra alcuna variazione dei parametri di vitalità cellulare e dei marcatori di inibizione del proteasoma e di UPR (rispetto a quelli osservati nel singolo trattamento con MG132). Essendo l’autofagia un meccanismo compensativo che si attiva in seguito all’inibizione del proteasoma o allo stress del RE, abbiamo valutato che ruolo potesse avere tale meccanismo nella risposta simil-antagonistica osservata in seguito al co-trattamento con EGCG e BZM. I nostri risultati hanno evidenziato, in cellule trattate con BZM, l’attivazione di un flusso autofagico che si intensifica quando viene addizionato l’EGCG. Tramite l’inibizione dell’autofagia mediante co-somministrazione di clorochina, è stato possibile stabilire che l’autofagia indotta dall’EGCG favorisce la sopravvivenza delle cellule sottoposte al trattamento combinato tramite la riduzione dell’UPR. Queste evidenze ci portano a concludere che per il trattamento del CaP è sconsigliabile associare le catechine del tè verde con il BZM e che in futuri studi di combinazione di questi polifenoli con composti antitumorali sarà importante valutare il ruolo dell’autofagia come possibile meccanismo di resistenza.

Potencial da ressonância magnética multiparamétrica na deteção de carcinoma da próstata localizado e valor do coeficiente de difusão aparente como possível preditor da agressividade tumoral : revisão de uma técnica em expansão

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Avaliação de resposta a Vinorelbina-Estramustina em segunda linha terapêutica, em doentes com carcinoma da próstata hormono-refratário previamente tratados com Docetaxel

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

The role of angiogenesis in prostate development and the pathogenesis of prostate cancer

New vessel formation, a highly-regulated, active process commencing in the embryo and evident notably during the pubertal growth spurt, is essential for normal prostate development. Reactivation of this process in response to physiological stimuli, particularly hypoxia in mature tissues, occurs with new vessels forming principally from stromal components. Although angiogenesis is complex, putatively involving a multitude of angiogenic factors and inhibitors, there is powerful evidence of the importance of the VEGF system in the development of both the normal prostate and prostate cancer. Recent advances include an understanding of how castration acts through the VEGF system to inhibit angiogenesis. Stromal-endothelial and epithelial-endothelial interactions are just beginning to be investigated. A better understanding of how physiological angiogenesis is controlled should help to provide further insights into the mechanism of disregulated angiogenesis in tumours. Ultimately, new antiangiogenic agents are likely to find a role in the management of patients with prostate cancer.

Acceptability of short term neo-adjuvant androgen deprivation in patients with locally advanced prostate cancer

Purpose: To determine the acceptability of short term neo-adjuvant maximal androgen deprivation (MAD) to patients treated with external beam radiation for locally advanced prostate cancer. Methods: Between 1996 and 2000, 818 patients with locally advanced, but non-metastatic, prostate cancer were entered into a randomised clinical trial (TROG 96.01), which compared radiation treatment alone with the same radiation treatment and 3 or 6 months neo-adjuvant MAD with goserelin and flutamide. Relevant symptoms, and how troublesome they were to the patient, were scored using a self-assessment questionnaire. This was completed by the patient at registration, and at specified times during and after treatment. Patients taking flutamide had liver function tests checked at regular intervals. Results: All patients have completed at least 12 months follow-up after treatment. Nearly all patients completed planned treatment with goserelin, but 27% of patients in the 6-month MAD treatment arm, and 20% in the 3-month arm, had to stop flutamide early. This was mainly due to altered liver function (up to 17% patients) and bowel side effects (up to 8% patients). However, although flutamide resulted in more bowel symptoms for patients on MAD, there was significant reduction in some urinary symptoms on this treatment. Acute bowel and urinary side effects at the end of radiation treatment were similar in all treatment arms. Side effect severity was unrelated to radiation target volume size, which was reduced by MAD, but symptomatology prior to any treatment was a powerful predictor. Of the 36% of patients who were sexually active before any treatment, the majority became inactive whilst on MAD. However, sexual activity at 12 months after radiation treatment was similar in all treatment arms, indicating that the effects of short term MAD on sexual function are reversible. Conclusion: Despite temporary effects on sexual activity, and compliance difficulties with flutamide, short-term neo-adjuvant MAD was not perceived by patients to be a major inconvenience. If neo-adjuvant MAD in the way tested can be demonstrated to lead to improved biochemical control and/or survival, then patients would view these therapeutic gains as worthwhile. Compliance with short-term goserelin was excellent, confirming that LH-RH analogues have a potential role in more long-term adjuvant treatment. However, for more protracted androgen deprivation, survival advantages and deleterious effects need to be assessed in parallel, in order to determine the optimal duration of treatment. (C) 2003 Elsevier Ireland Ltd. All fights reserved.

Prospective study of men's psychological and decision-related adjustment after treatment for localized prostate cancer

Objectives. To undertake a prospective longitudinal study to assess psychological and decision-related distress after the diagnosis of localized prostate cancer. Methods. A total of I 11 men (93% response rate) with localized prostate cancer were recruited from outpatient urology clinics and urologists' private practices. More than one half (56%) elected to undergo radical prostatectomy, 19% underwent external beam radiotherapy, and 25% chose watchful waiting. Men completed self-report measures before treatment and 2 and 12 months after treatment. The measures used included the University of California, Los Angeles, Prostate Cancer Index, International Prostate Symptom Score, Impact of Events Scale, Constructed Meaning Scale, Satisfaction with Life Scale, Health Care Orientation subscale, and Decisional Conflict Scale. Results. No statistically significant differences were found by medical treatment group in the psychological and decision-related adjustment at baseline or with time. Men who were undecided about their treatment choice had greater decisional conflict and a more negative healthcare orientation, but were not more psychologically distressed, compared with men who had decided. At diagnosis, 63% of men had high decision-related distress, and this persisted for 42% of men 12 months after treatment, despite high satisfaction with their treatment choice. At diagnosis, low-to-moderate psychological distress was most common, with distress decreasing after treatment. The overall quality of life was similar to community norms. Conclusions. The results of our study indicated that men who were undecided about what treatment to receive experienced greater decision-related distress. The final treatment choice was not related to psychological distress about prostate cancer. Psychological and decision-related distress decreased with time, independent of treatment modality. Interventions should target decision-related distress for all men and in-depth psychological support for those who experience ongoing difficulties. (C) 2004 Elsevier Inc.

Resistance training and reduction of treatment side effects in prostate cancer patients

Purpose: To examine the effect of progressive resistance training on muscle function, functional performance, balance, body composition, and muscle thickness in men receiving androgen deprivation for prostate cancer. Methods: Ten men aged 59-82 yr on androgen deprivation for localized prostate cancer undertook progressive resistance training for 20 wk at 6- to 12-repetition maximum (RM) for 12 upper- and lower-body exercises in a university exercise rehabilitation clinic. Outcome measures included muscle strength and muscle endurance for the upper and lower body, functional performance (repeated chair rise, usual and fast 6-m walk, 6-m backwards walk, stair climb, and 400-m walk time), and balance by sensory organization test. Body composition was measured by dual-energy x-ray absorptiometry and muscle thickness at four anatomical sites by B-mode ultrasound. Blood samples were assessed for prostate specific antigen (PSA), testosterone, growth hormone (GH), cortisol, and hemoglobin. Results: Muscle strength (chest press, 40.5%; seated row, 41.9%; leg press, 96.3%; P < 0.001) and muscle endurance (chest press, 114.9%; leg press, 167.1%; P < 0.001) increased significantly after training. Significant improvement (P < 0.05) occurred in the 6-m usual walk (14.1%), 6-m backwards walk (22.3%), chair rise (26.8%), stair climbing (10.4%), 400-m walk (7.4%), and balance (7.8%). Muscle thickness increased (P < 0.05) by 15.7% at the quadriceps site. Whole-body lean mass was preserved with no change in fat mass. There were no significant changes in PSA, testosterone, GH, cortisol, or hemoglobin. Conclusions: Progressive resistance exercise has beneficial effects on muscle strength, functional performance and balance in older men receiving androgen deprivation for prostate cancer and should be considered to preserve body composition and reduce treatment side effects.

JNK regulates Fas receptor mediated apoptosis in prostate cancer cell lines

Prostate Cancer is a disease that primarily affects elderly men. The incidence of prostate cancer has been progressively increasing in the western world over the last two decades. Life expectancy and diet are believed to be the main factors contributing to this increase in prevalence. Prostate cancer is a slowly progressing disorder and patients often live for over 10 years after initially being diagnosed with prostate cancer. However, patients with hormone refractory prostate cancer have a poor prognosis and generally do not survive for longer than 2 or 3 years. Hormone refractory prostate cancer is responsible for over 200,000 deaths each year and current chemotherapeutic regimens are only useful as palliative agents. The long-term survival rate is poor and chemotherapy does not significantly increase this. Cell lines derived from hormone refractory tumours usually display elevated resistance to many cytotoxic drugs. The Fas receptor is a membrane bound protein capable of binding to a ligand called Fas ligand. Engagement of Fas receptor with Fas ligand results in clustering of Fas receptor on the plasma membrane of cells. A number of proteins responsible for initiating apoptosis are recruited to the plasma membrane and are activated in response to elevated local concentrations. This series of events initiates a proteolysis cascade and that culminates in the degradation of structural and enzymatic processes and the repackaging of cellular constituents within membrane bound vesicles that can be endocytosed and recycled by surrounding phagocytic cells. The Fas receptor is believed to be a key mechanism by which immune cells can destroy damaged cells. Consequently, resistance to Fas receptor mediated apoptosis often correlates with tumour progression. It has been reported that prostate cancer cell lines display elevated resistance to Fas receptor mediated apoptosis and this correlates with the stage of tumour from which the cell lines were isolated. JNK, a stress-activated protein kinase, has been implicated both with increased survival and increased apoptosis in prostate cancer. Elevated endogenous JNK activity has been demonstrated to correlate with prostate cancer progression. It has been shown that endogenous JNK activity increases the expression of anti-apoptotic proteins and can increase the resistance of prostate cancer cell lines to chemotherapy. In addition, elevated endogenous JNK activity is required for improved proliferation and transformation of a number of epithelial tumours. However, prolonged JNK activation in response to cytotoxic stimuli can increase the sensitivity of cells to apoptosis. Prolonged JNK activity appears to induce the expression of a separate set of genes responsible for promoting apoptosis. Our group has recently shown that activation of JNK by chemotherapeutic drugs can sensitise DU 145 prostate carcinoma cells to Fas receptor mediated apoptosis. In order toidentify novel targets for treating hormone refractory prostate cancer we have investigated the role of JNK in Fas receptor mediated apoptosis. We have demonstrated that prolonged JNK activation is defective in DU 145 cells in response to Fas receptor activation alone. Co-administering anisomycin, a JNK agonist, greatly enhances the ability of DU 145 cells to undergo apoptosis by increasing the rate of Caspase 8 cleavage. We also investigated the role of endogenous JNK activity in Fas receptor mediated.

Glycosylation is an Androgen-Regulated Process Essential for Prostate Cancer Cell Viability

Steroid androgen hormones play a key role in the progression and treatment of prostate cancer, with androgen deprivation therapy being the first-line treatment used to control cancer growth. Here we apply a novel search strategy to identify androgen-regulated cellular pathways that may be clinically important in prostate cancer. Using RNASeq data, we searched for genes that showed reciprocal changes in expression in response to acute androgen stimulation in culture, and androgen deprivation in patients with prostate cancer. Amongst 700 genes displaying reciprocal expression patterns we observed a significant enrichment in the cellular process glycosylation. Of 31 reciprocally-regulated glycosylation enzymes, a set of 8 (GALNT7, ST6GalNAc1, GCNT1, UAP1, PGM3, CSGALNACT1, ST6GAL1 and EDEM3) were significantly up-regulated in clinical prostate carcinoma. Androgen exposure stimulated synthesis of glycan structures downstream of this core set of regulated enzymes including sialyl-Tn (sTn), sialyl Lewis(X) (SLe(X)), O-GlcNAc and chondroitin sulphate, suggesting androgen regulation of the core set of enzymes controls key steps in glycan synthesis. Expression of each of these enzymes also contributed to prostate cancer cell viability. This study identifies glycosylation as a global target for androgen control, and suggests loss of specific glycosylation enzymes might contribute to tumour regression following androgen depletion therapy.

Carcinoma of unknown primary (CUP); some considerations about pathogenesis and diagnostic strategy, particularly focusing on CUPS pertaining to the Urology

he term "carcinoma of unknown primary" (CUP) defines a malignant condition in which a metastatic cancer is documented in absence of a detectable primary site. It occurs in about 2÷6 % of cancer patients, according to various literature reports. The primary tumor site results indefinable because of several either single or associated factors, even remaining occult at autopsy in 15÷25% of CUP patients. The metastatic spread pattern of CUP is quite unlike that expected for analogous known primary malignancy. For instance, the unknown prostate cancer often metastasizes to the lungs and liver while the its known analogous usually spreads to the bone. Whether certain genetic abnormalities might play a role in determining a CUP condition, it remains undefined. Most CUP are adenocarcinoma, squamous cell carcinoma, either undifferentiated or differentiated carcinoma, whereas less frequently may be sarcoma, melanoma or neuroendocrine tumor. As CUP diagnostic management is concerned, two opposite approach modalities may be adopted, one, named "shotgun modality", consisting in a multiplicity of examinations aimed at achieving the identification of the primary tumor and the other, a nihilistic modality, by adopting tout court a palliative therapy of the metastatic disease. A reasonable intermediate diagnostic strategy consists in undertaking some procedures with a specific target and low cost/benefit ratio. Selected imaging studies, serum tumor markers, immunohistochemical analyses and genetic- molecular examinations on biopsy material allow sometimes to reach the detection of primary malignancies that might be responsive to a potential treatments. Nevertheless, in spite of recent sophisticated -laboratory and imaging progress, CUP remains a strong challenge in clinical oncology.

Generation and Characterisation of a Canine EGFP-HMGA2 Prostate Cancer In Vitro Model

The architectural transcription factor HMGA2 is abundantly expressed during embryonic development. In several malignant neoplasias including prostate cancer, high re-expression of HMGA2 is correlated with malignancy and poor prognosis. The let-7 miRNA family is described to regulate HMGA2 negatively. The balance of let-7 and HMGA2 is discussed to play a major role in tumour aetiology. To further analyse the role of HMGA2 in prostate cancer a stable and highly reproducible in vitro model system is precondition. Herein we established a canine CT1258-EGFP-HMGA2 prostate cancer cell line stably overexpressing HMGA2 linked to EGFP and in addition the reference cell line CT1258-EGFP expressing solely EGFP to exclude EGFP-induced effects. Both recombinant cell lines were characterised by fluorescence microscopy, flow cytometry and immunocytochemistry. The proliferative effect of ectopically overexpressed HMGA2 was determined via BrdU assays. Comparative karyotyping of the derived and the initial CT1258 cell lines was performed to analyse chromosome consistency. The impact of the ectopic HMGA2 expression on its regulator let-7a was analysed by quantitative real-time PCR. Fluorescence microscopy and immunocytochemistry detected successful expression of the EGFP-HMGA2 fusion protein exclusively accumulating in the nucleus. Gene expression analyses confirmed HMGA2 overexpression in CT1258-EGFP-HMGA2 in comparison to CT1258-EGFP and native cells. Significantly higher let-7a expression levels were found in CT1258-EGFP-HMGA2 and CT1258-EGFP. The BrdU assays detected an increased proliferation of CT1258-HMGA2-EGFP cells compared to CT1258-EGFP and native CT1258. The cytogenetic analyses of CT1258-EGFP and CT1258-EGFP-HMGA2 resulted in a comparable hyperdiploid karyotype as described for native CT1258 cells. To further investigate the impact of recombinant overexpressed HMGA2 on CT1258 cells, other selected targets described to underlie HMGA2 regulation were screened in addition. The new fluorescent CT1258-EGFP-HMGA2 cell line is a stable tool enabling in vitro and in vivo analyses of the HMGA2-mediated effects on cells and the development and pathogenesis of prostate cancer.