Growth hormone-releasing hormone: An autocrine growth factor for small cell lung carcinoma

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various cancers in vivo. This effect is thought to be exerted through suppression of the pituitary growth hormone–hepatic insulin-like growth factor I (IGF-I) axis and direct inhibition of autocrine/paracrine production of IGF-I and -II in tumors. However, other evidence points to a direct effect of GHRH antagonists on tumor growth that may not implicate IGFs, although an involvement of GHRH in the proliferation of cancer cells has not yet been established. In the present study we investigated whether GHRH can function as an autocrine/paracrine growth factor in small cell lung carcinoma (SCLC). H-69 and H-510A SCLC lines cultured in vitro express mRNA for GHRH, which apparently is translated into peptide GHRH and then secreted by the cells, as shown by the detection of GHRH-like immunoreactivity in conditioned media from the cells cultured in vitro. In addition, the levels of GHRH-like immunoreactivity in serum from nude mice bearing H-69 xenografts were higher than in tumor-free mice. GHRH(1–29)NH2 stimulated the proliferation of H-69 and H-510A SCLCs in vitro, and GHRH antagonist JV-1–36 inhibited it. JV-1–36 administered s.c. into nude mice bearing xenografts of H-69 SCLC reduced significantly (P < 0.05) tumor volume and weight, after 31 days of therapy, as compared with controls. Collectively, our results suggest that GHRH can function as an autocrine growth factor in SCLCs. Treatment with antagonistic analogs of GHRH may offer a new approach to the treatment of SCLC and other cancers.

Human renal cell carcinoma expresses distinct binding sites for growth hormone-releasing hormone

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the proliferation of various human cancers in vitro and in vivo by mechanisms that include apparent direct effects through specific binding sites expressed on tumors and that differ from pituitary human GHRH (hGHRH) receptors. In this study, GHRH antagonist JV-1–38 (20 μg/day per animal s.c.) inhibited the growth of orthotopic CAKI-1 human renal cell carcinoma (RCC) by 83% and inhibited the development of metastases to lung and lymph nodes. Using ligand competition assays with 125I-labeled GHRH antagonist JV-1–42, we demonstrated the presence of specific high-affinity (Kd = 0.25 ± 0.03 nM) binding sites for GHRH with a maximal binding capacity (Bmax) of 70.2 ± 4.1 fmol/mg of membrane protein in CAKI-1 tumors. These receptors bind GHRH antagonists preferentially and display a lower affinity for hGHRH. The binding of 125I-JV-1–42 is not inhibited by vasoactive intestinal peptide (VIP)-related peptides sharing structural homology with hGHRH. The receptors for GHRH antagonists on CAKI-1 tumors are distinct from binding sites detected with 125I-VIP (Kd = 0.89 ± 0.14 nM; Bmax = 183.5 ± 2.6 fmol/mg of protein) and also have different characteristics from GHRH receptors on rat pituitary as documented by the insignificant binding of [His1,125I-Tyr10,Nle27]hGHRH(1–32)NH2. Reverse transcription-PCR revealed the expression of splice variants of hGHRH receptor in CAKI-1 RCC. Biodistribution studies demonstrate an in vivo uptake of 125I-JV-1–42 by the RCC tumor tissue. The presence of specific receptor proteins that bind GHRH antagonists in CAKI-1 RCC supports the view that distinct binding sites that mediate the inhibitory effect of GHRH antagonists are present on various human cancers.

Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers

The proliferation of various tumors is inhibited by the antagonists of growth hormone-releasing hormone (GHRH) in vitro and in vivo, but the receptors mediating the effects of GHRH antagonists have not been identified so far. Using an approach based on PCR, we detected two major splice variants (SVs) of mRNA for human GHRH receptor (GHRH-R) in human cancer cell lines, including LNCaP prostatic, MiaPaCa-2 pancreatic, MDA-MB-468 breast, OV-1063 ovarian, and H-69 small-cell lung carcinomas. In addition, high-affinity, low-capacity binding sites for GHRH antagonists were found on the membranes of cancer cell lines such as MiaPaCa-2 that are negative for the vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide receptor (VPAC-R) or lines such as LNCaP that are positive for VPAC-R. Sequence analysis of cDNAs revealed that the first three exons in SV1 and SV2 are replaced by a fragment of retained intron 3 having a new putative in-frame start codon. The rest of the coding region of SV1 is identical to that of human pituitary GHRH-R, whereas in SV2 exon 7 is spliced out, resulting in a 1-nt upstream frameshift, which leads to a premature stop codon in exon 8. The intronic sequence may encode a distinct 25-aa fragment of the N-terminal extracellular domain, which could serve as a proposed signal peptide. The continuation of the deduced protein sequence coded by exons 4–13 in SV1 is identical to that of pituitary GHRH-R. SV2 may encode a GHRH-R isoform truncated after the second transmembrane domain. Thus SVs of GHRH-Rs have now been identified in human extrapituitary cells. The findings support the view that distinct receptors are expressed on human cancer cells, which may mediate the antiproliferative effect of GHRH antagonists.