Pituitary adenylate cyclase-activating peptide/vasoactive intestinal peptide receptors in human normal mammary gland and breast cancer tissue.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) bind similarly to VPAC1 and VPAC2 receptors, whereas PACAP binds with higher affinity than VIP to PAC1 receptors. Here we demonstrate by different approaches the expression of the three subclasses of PACAP/VIP receptors in human normal and malignant breast tissue. At the mRNA level, reverse transcription-polymerase chain reaction experiments showed VPAC1 and VPAC2 receptors as well as various isoforms (null, hip/hop) of PAC1 receptors due to alternative splicing. At the protein level, Western blot experiments revealed the three subclasses of receptor although no conclusive differences could be established when comparing control, peritumoral and tumoral tissue samples. Immunohistochemistry showed the distribution of these receptors: they were located at epithelial cells in normal and cancer conditions but also in leukocytes at the stromal level in carcinomatous tissue. A weaker immunostaining of PAC1 receptors in normal tissue and a strong density of the three PACAP/VIP receptor subclasses in cancer tissue may be related to differential expression patterns during breast tumor progression but more samples need to be studied to validate this hypothesis. PAC1, VPAC1 and VPAC2 receptors were functional, as shown by their coupling to adenylate cyclase stimulation: VIP, PACAP-27 and PACAP-38 behaved similarly at this level, whereas both VPAC receptors acted alike as shown by means of specific peptide agonists and antagonists. The present results together with the known presence of PACAP and VIP in the mammary gland support a paracrine/autocrine involvement of both peptides at this level in physiological and pathological conditions, i.e. during malignant transformation.

PACAP expression and distribution in human breast cancer and healthy tissue.

Pituitary adenylate cyclase-activating peptide (PACAP) is involved in various biological processes including cell growth, proliferation and differentiation. Here, we demonstrate for the first time the presence of both PACAP mRNA and PACAP immunoreactivity in human normal breast and breast carcinoma. Control, peritumoral and tumoral tissue pieces expressed preproPACAP mRNA since reverse transcription-polymerase chain reaction analysis gave an amplification product of the expected size (226 bp), which was further identified by analysis of the sequence. A main 19.9-kDa product (preproPACAP protein) was identified by Western blot in the three classes of breast tissue together with a small amount of a 14.6-kDa product (conceivably a result of partial processing by proprotein convertases). However, the mature peptide PACAP-38 was absent. The levels of both PACAP mRNA and PACAP immunoreactivity increased from normal to peritumoral and tumoral breast tissues but more patients must be considered to reinforce this feature. Immunohistochemistry showed the localization of PACAP immunoreactivity in alveolar epithelial cells in normal and carcinomatous tissues but also, at high density, in duct-like structures and in leucocytes in the connective tissue from breast cancer pieces. The results suggest that PACAP may play a role by autocrine/paracrine mechanisms in both normal human breast physiology and breast tumorigenesis.

Expression of functional PACAP/VIP receptors in human prostate cancer and healthy tissue.

Vasoactive intestinal peptide (VIP) is involved in prostate cell proliferation and function. VIP and pituitary adenylate cyclase-activating peptide (PACAP) are similarly recognized by VPAC(1)/VPAC(2) receptors whereas PACAP binds with higher affinity than VIP to PAC(1) receptor. Here we systematically studied the presence and distribution of functional PAC(1), VPAC(1) and VPAC(2) receptors in human normal and malignant prostate tissue. Functional PACAP/VIP receptors were detected in normal and malignant prostate by adenylyl cyclase stimulation with PACAP-27/38 and VIP. RT-PCR experiments showed PAC(1) (various isoforms due to alternative splicing), VPAC(1) and VPAC(2) receptor expression at the mRNA level, whereas Western blots found the three receptor protein classes in normal and pathological conditions. No conclusive differences could be established when comparing control and cancer tissue samples. Immunohistochemistry showed a weaker immunostaining in tumoral than in normal epithelial cells for the three receptor subtypes. In conclusion, we demonstrate the expression of functional PAC(1), VPAC(1) and VPAC(2) receptors in human prostate as well as its maintenance after malignant transformation.

The expression of growth hormone-releasing hormone (GHRH) and its receptor splice variants in human breast cancer lines; the evaluation of signaling mechanisms in the stimulation of cell proliferation.

Antagonists of growth hormone-releasing hormone (GHRH) inhibit growth of various human cancers including breast cancer, xenografted into nude mice or cultured in vitro. Splice variants (SVs) of receptors for GHRH have been found in several human cancers and cancer cell lines. The antiproliferative actions of GHRH antagonists could be mediated in part through these SVs of GHRH receptors. In this study we examined the expression of mRNA for GHRH and SVs of its receptors in human breast cancer cell lines MCF-7, MCF-7MIII, MDA-MB-231, MDA-MB-435, MDA-MB-468, and T47D. mRNA for GHRH was present in all lines tested. mRNA for SV1 isoform of GHRH receptors was found in MCF-7MIII, MDA-MB-468, and T47D; and for SV2 isoform in MCF-7MIII and T47D cell lines. In proliferation studies in vitro, the growth of T47D cells was stimulated by GHRH and dose-dependently inhibited by GHRH antagonist JV-1-38. H89 (protein kinase A inhibitor), bisindolylmaleimide I (protein kinase C [PKC] inhibitor) and verapamil (voltage-dependent calcium channel blocker) inhibited the GHRH-stimulated proliferation of T47D cells. The GHRH antagonist JV-1-38 suppressed the T47D cell growth in vitro stimulated by PKC activator (phorbol-12-myristate-13-acetate). The stimulation of T47D cells by GHRH was followed by an increase in cAMP production and GHRH antagonist JV-1-38 competitively inhibited this effect. Our results suggest that SVs of GHRH receptors could mediate the responses to GHRH and GHRH antagonists in breast cancer through Ca2+-, cAMP- and PKC-dependent mechanisms. The presence of SV1 of GHRH receptors in human cancers provides a rationale for antitumor therapy based on the blockade of this receptor by specific GHRH antagonists.

Expression and distribution of pituitary adenylate cyclase-activating peptide in human prostate and prostate cancer tissues.

The presence, expression and distribution of pituitary adenylate cyclase-activating peptide (PACAP) in human prostate cancer and healthy tissue were investigated by means of biochemical and morphological procedures. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated the presence of its precursor encoding mRNA in both normal and pathological conditions (amplification products with 577 or 226 bp were identified). Immunochemistry using an appropriate antibody served to detect in both classes of tissues a 19.9-kDa product corresponding to the PACAP preproprotein and another protein of 14.6 kDa that may represent a product partially processed by convertases. However, a 5-kDa band characteristic of PACAP-38 peptide was not observed. Immunohistochemistry on tissue sections indicated the location of PACAP in the epithelial layer of prostate glands (and in some scarce leucocytes) but not in the stroma, either in normal or carcinomatous tissues. No clear differences could be established when comparing samples from patients with different tumor Gleason grades. These results are the first demonstration of the localization of PACAP or its precursors and its mRNA in the human prostate gland and their presence during the progression of prostate carcinoma.

The expression of growth hormone-releasing hormone (GHRH) and splice variants of its receptor in human gastroenteropancreatic carcinomas.

Splice variants (SVs) of receptors for growth hormone-releasing hormone (GHRH) have been found in primary human prostate cancers and diverse human cancer cell lines. GHRH antagonists inhibit growth of various experimental human cancers, including pancreatic and colorectal, xenografted into nude mice or cultured in vitro, and their antiproliferative action could be mediated in part through SVs of GHRH receptors. In this study we examined the expression of mRNA for GHRH and for SVs of its receptors in tumors of human pancreatic, colorectal, and gastric cancer cell lines grown in nude mice. mRNA for both GHRH and SV(1) isoform of GHRH receptors was expressed in tumors of pancreatic (SW1990, PANC-1, MIA PaCa-2, Capan-1, Capan-2, and CFPAC1), colonic (COLO 320DM and HT-29), and gastric (NCI-N87, HS746T, and AGS) cancer cell lines; mRNA for SV(2) was also present in Capan-1, Capan-2, CFPAC1, HT-29, and NCI-N87 tumors. In proliferation studies in vitro, the growth of pancreatic, colonic, and gastric cancer cells was stimulated by GHRH(1-29)NH(2) and inhibited by GHRH antagonist JV-1-38. The stimulation of some gastroenteropancreatic cancer cells by GHRH was followed by an increase in cAMP production, and GHRH antagonist JV-1-38 competitively inhibited this effect. Our study indicates the presence of an autocrine/paracrine stimulatory loop based on GHRH and SV(1) of GHRH receptors in human pancreatic, colorectal, and gastric cancers. The finding of SV(1) receptor in human cancers provides an approach to an antitumor therapy based on the blockade of this receptor by specific GHRH antagonists.