Malignant mesothelioma is a devastating disease that has been associated with loss of Neurofibromatosis type 2 (NF2) and genetic lesions affecting RB and P53 pathways. We introduced similar lesions in the mesothelial lining of the thoracic cavity of mice. Mesothelioma developed at high incidence in Nf2;Ink4a/Arf and Nf2;p53 conditional knockout mice with median survival times of approximately 30 and 20 weeks, respectively. Murine mesothelioma closely mimicked human malignant mesothelioma. Conditional Nf2;Ink4a/Arf mice showed increased pleural invasion compared to conditional Nf2;p53 mice. Interestingly, upon Ink4a loss in the latter mice median survival was significantly reduced and all tumors were highly invasive, suggesting that Ink4a loss substantially contributes to the poor clinical outcome of malignant mesothelioma.

BACKGROUND Androgen‐independent growth leads to progressive prostate cancer after androgen‐ablation therapy. This may be caused by altered specificity of the androgen receptor (AR), by ligand‐independent stimulation of the AR, or by paracrine growth modulation by neuropeptides secreted by neuroendocrine (NE) cells. METHODS We established and characterized the androgen‐independent FGC‐DCC from the androgen‐dependent LNCaP fast growing colony (FGC) cell line. The androgen‐independent DU‐145, FGC‐DCC, and PC‐3, and the androgen‐dependent LNCaP and PC‐346C cell lines were used to study growth modulation of gastrin‐releasing peptide (GRP), calcitonin (CT), serotonin (5‐HT), and vasoactive intestinal peptide (VIP) by 3H‐thymidine incorporation. Specificity of the growth‐modulating effects was tested with the anti‐GRP monoclonal antibody 2A11 and induction of cAMP by neuropeptides. RESULTS Androgen‐independent growth stimulation by neuropeptides was shown in DU‐145 and PC‐346C. 2A11 inhibited GRP‐induced 3H‐thymidine incorporation in DU‐145 and PC‐346C and inhibited proliferation of the FGC‐DCC and PC‐3 cell lines. With some exceptions, cAMP induction paralleled growth stimulation. Dideoxyadenosine (DDA) inhibited the GRP‐induced growth effect in DU‐145 and PC‐346C, whereas oxadiazoloquinoxaline‐1‐one (ODQ) had no effect on 3H‐thymidine incorporation. None of the neuropeptides stimulated growth of LNCaP, FGC‐DCC, or PC‐3. CONCLUSIONS GRP‐induced growth of DU‐145 and PC‐346C was specific and cAMP‐mediated. Androgen‐independent growth of FGC‐DCC cells was mainly due to an induction of Bcl‐2 expression and possibly through the activation of an autocrine and NE‐like pathway, as has been shown also for the PC‐3 cell line. Growth induction of non‐NE cells by neuropeptides could be a possible role for NE cells in clinical prostate cancer. Prostate 42:34–44, 2000. © 2000 Wiley‐Liss, Inc.

It was previously shown in the PC-295 xenograft that the number of chromogranin A (CgA)-positive neuroendocrine (NE) cells increased after androgen withdrawal. NE cells did not proliferate and differentiated from G 0 -phase-arrested cells.Here we further characterized NE differentiation , androgen receptor status, and apoptosis-associated Bcl-2 expression in the PC-295 model after androgen withdrawal to assess the origin of NE cells. PC-295 tumor volumes decreased by 50% in 4 days. Intraperitoneal bromodeoxyuridine (BrdU) incorporation and MIB-1 labeling decreased to 0% , and the apoptosis was maximal at day 4. Androgen receptor expression and prostate-specific antigen (PSA) serum levels decreased rapidly within 2 days. The number of NE cells increased 6-fold at day 4 and 30-fold at day 7. Five and ten percent of the CgApositive cells were BrdU positive after continuous BrdU labeling for 2 and 4 days , respectively. However , no MIB-1 expression was observed in CgA-positive cells. NE cells expressed the regulated secretory pathway marker secretogranin III but were negative for androgen receptor and Bcl-2. Bcl-2 expression did increase in the non-NE tumor cells. In conclusion, androgen withdrawal leads to a rapid PC-295 tumor regression and a proliferation-independent induction of NE differentiation. The strictly androgen-independent NE cells that were still present after 21 days differentiated mainly from G 0 -phase-arrested cells. 2-8 These cells produce various growth-modulating neuropeptides in a paracrine or autocrine way. -15Possible roles for NE cells in the prostate may be regulation of homeostasis and secretion of prostatic fluid, either actively or passively. NE cells can be identified by routine electron microscopy (dense core granules) or by immunohistochemistry with specific antibodies against secreted products, for example, serotonin, 16 or secretion-associated proteins, such as chromogranin A (CgA), [17][18][19] which is a marker for neuroendocrine differentiation. NE cells are considered to be nonproliferating cells and do not express the androgen receptor 20 and are therefore probably unaffected by androgen deprivation. Consequently, they will not undergo apoptosis under such circumstances. Therefore, it is relevant to assess whether or not CgA-positive cells co-express the antiapoptotic oncogene Bcl-2. 21 Moreover, NE cells show a heterogeneous cytokeratin expression pattern as there are basal, luminal, and intermediate NE cell types 16,22,23 and are often found near Bcl-2-positive prostate cancer cells. 24,25The single expression of CgA is not the only requisite for the determination of NE differentiated cells as there exists a regulated secretory pathway (RSP) in NE cells 26 next to the lysosomal and an exocrine constitutive pathway. Along the RSP pathway, secretion and processing of bioactive neuropeptides and growth hormones, such as insulin and glucagon in the pancreas, 27,28 are regulated. The RSP consists of a sequence of processes linked from transcription/translation of various factors ...

Most PCs show NE differentiation. Several studies have tried to correlate NE expression with disease status, but the reported findings have been contradictory. Prostatic NE cells synthesize peptides with a wide spectrum of potential functions. Some of these active peptides, such as PAMP, are amidated. PAM is the only carboxy-terminal peptide-amidating enzyme identified. We studied expression of PAMP and PAM in normal prostate and prostatic tumors (clinical specimens and human xenograft models) with or without prior androgen-deprivation therapy and found a wide distribution of both molecules in NE subpopulations of all kinds. Although the correlation of either marker to tumor grade, clinical progression or disease prognosis did not reach statistical significance, PAMP-or PAM-immunoreactive cells were induced after androgen-blockade therapy. In the PC-310 and PC-295 androgen-dependent models, PAMP or PAM NE differentiation was induced after castration in different ways, being higher in PC-310, which might explain its long-term survival after androgen deprivation. We show induction of expression of 2 new NE markers in clinical specimens and xenografted PC after endocrine therapy.