Loss of transformed phenotype in cancer cells by overexpression of the uteroglobin gene

Abstract

Uteroglobin (UG) is a multifunctional, secreted protein that has receptor-mediated functions. The human UG (hUG) gene is mapped to chromosome 11q12.2-13.1, a region frequently rearranged or deleted in many cancers. Although high levels of hUG expression are characteristic of the mucosal epithelia of many organs, hUG expression is either drastically reduced or totally absent in adenocarcinomas and in viral-transformed epithelial cells derived from the same organs. In agreement with these findings, in an ongoing study to evaluate the effects of aging on UG-knockout mice, 16/16 animals developed malignant tumors, whereas the wild-type littermates (n = 25) remained apparently healthy even after 11/2 years. In the present investigation, we sought to determine the effects of induced-expression of hUG in human cancer cells by transfecting several cell lines derived from adenocarcinomas of various organs with an hUG-cDNA construct. We demonstrate that induced hUG expression reverses at least two of the most important characteristics of the transformed phenotype (i.e., anchorage-independent growth on soft agar and extracellular matrix invasion) of only those cancer cells that also express the hUG receptor. Similarly, treatment of the nontransfected, receptor-positive adenocarcinoma cells with purified recombinant hUG yielded identical results. Taken together, these data define receptor-mediated, autocrine and paracrine pathways through which hUG reverses the transformed phenotype of cancer cells and consequently, may have tumor suppressor-like effects.

Figure 1

(A) RT-PCR analysis of total RNA extracted from pRC/RSV-hUG-transfected and wild-type adenocarcinomas of the uterus (HEC-1A) and the prostate (HTB-81). The PCR products were blotted and detected by hybridization with a hUG-specific oligonucleotide probe, hUGP (21). Amplification of the human GADPH gene was used as an internal control for RNA quality and to rule out pipeting error. Lanes 1 and 2 represent two independently derived pRC/RSV-hUG-transfected clones each of HEC-1A (Left) and HTB-81 (Right), respectively. Wt, wild-type (nontransfected) cells. (B) Western blot analyses showing UG production by HEC-1A and HTB-81 cells transfected with pRC/RSV-hUG. Proteins were resolved by SDS/PAGE under reducing and denaturing conditions (see Materials and Methods for details). UG(d), UG-dimer; UG(m), UG-monomer.

Figure 2

Anchorage-independent growth on soft agar. (A) HEC-1A cells that were not transfected (wild type). (B) HEC-1A cells that were pRC/RSV (mock)-transfected. (C) HEC-1A cells transfected with pRC/RSV-hUG. Note the striking suppression of anchorage-independent growth of pRC/RSV-hUG-transfected cells (C). Magnification: ×20.

Figure 3

Effect of the expression of hUG on ECM invasion by HEC-1A cells. (A) Wild-type (nontransfected) cells. (B) Cells transfected with pRC/RSV-hUG expression construct. (C) Wild-type cells that were treated with pure hUG. Note the striking suppression of ECM invasion in B and C. Magnification: ×200. (D) Graphic representation of the quantitative inhibition of ECM invasion.

Figure 4

Scatchard analysis of specific binding of reduced ¹²⁵I-UG of wild-type HEC-1A cells (A). Affinity-crosslinking of ¹²⁵I-hUG with HEC-1A (B) and HTB-81 (C) cells. The cells were incubated with reduced ¹²⁵I-hUG in the absence or presence of unlabeled reduced hUG for binding and then crosslinked with DSS. Lane 1: (−)DSS; lane 2: (+)DSS, and lane 3: (+)hUG + DSS. Ori, origin. Note the complete absence of the hUG receptor(s) on HTB-81 cells.