LRIG1 opposes epithelial-to-mesenchymal transition and inhibits invasion of basal-like breast cancer cells.

LRIG1 (leucine-rich repeat and immunoglobulin-like domain containing), a member of the LRIG family of transmembrane leucine-rich repeat-containing proteins, is a negative regulator of receptor tyrosine kinase signaling and a tumor suppressor. LRIG1 expression is broadly decreased in human cancer and in breast cancer and low expression of LRIG1 has been linked to decreased relapse-free survival. Recently, low expression of LRIG1 was revealed to be an independent risk factor for breast cancer metastasis and death. These findings suggest that LRIG1 may oppose breast cancer cell motility and invasion, cellular processes that are fundamental to metastasis. However, very little is known of LRIG1 function in this regard. In this study, we demonstrate that LRIG1 is downregulated during epithelial-to-mesenchymal transition (EMT) of human mammary epithelial cells, suggesting that LRIG1 expression may represent a barrier to EMT. Indeed, depletion of endogenous LRIG1 in human mammary epithelial cells expands the stem cell population, augments mammosphere formation and accelerates EMT. Conversely, expression of LRIG1 in highly invasive Basal B breast cancer cells provokes a mesenchymal-to-epithelial transition accompanied by a dramatic suppression of tumorsphere formation and a striking loss of invasive growth in three-dimensional culture. LRIG1 expression perturbs multiple signaling pathways and represses markers and effectors of the mesenchymal state. Furthermore, LRIG1 expression in MDA-MB-231 breast cancer cells significantly slows their growth as tumors, providing the first in vivo evidence that LRIG1 functions as a growth suppressor in breast cancer.

Prospective randomised evaluation of traditional Chinese medicine combined with chemotherapy: a randomised phase II study of wild toad extract plus gemcitabine in patients with advanced pancreatic adenocarcinomas.

BACKGROUND: An intravenous formulated extract of the venom of the wild toad Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider, huachansu, is currently used in China for the treatment of lung, liver, pancreatic, and colorectal cancers. We performed a randomised, single-blinded, phase II clinical study of huachansu plus gemcitabine versus placebo plus gemcitabine in patients with locally advanced and/or metastatic pancreatic adenocarcinomas. METHODS: Patients with tissue-proven locally advanced and/or metastatic pancreatic adenocarinoma were randomly assigned to receive either gemcitabine 1000 mg m(-2) on days 1, 8, and 15 with huachansu 20 ml m(-2) daily for 21 days (arm A) or placebo (arm B); treatment cycles were 28 days in length. Primary end point was 4-month progression-free overall survival (PFS); secondary end points were objective radiographical response rate (ORR), time to progression (TTP), and toxicity. RESULTS: A total of 80 subjects were enrolled; 76 patients were evaluable (received at least 1 week therapy). Median overall survival was 160 days for arm A and 156 days for arm B (P=0.339); ORR was 9 and 3% in arms A and B, respectively (P=0.332), median TTP was 98 and 115 days, respectively (P=0.825); the median 4-month PFS was 99 and 98 days, respectively (P=0.679). CONCLUSION: Huachansu when combined with gemcitabine did not improve the outcome of patients with locally advanced and/or metastatic pancreatic cancer.

Phase I trial of capecitabine in combination with interferon alpha in patients with metastatic renal cancer: toxicity and pharmacokinetics.

PURPOSE: The present study was designed to determine the toxicity and maximum tolerated doses of oral intermittent oral capecitabine and subcutaneous (s.c.) rHuIFNalpha2a in patients with metastatic renal cell carcinoma (RCC). The pharmacokinetics of capecitabine and its metabolites were also investigated. METHODS: A total of 27 patients were treated at four dose levels of capecitabine (825 or 1000 mg/m2 twice daily orally, days 1-14, 22-36) and rHuIFNalpha2a (1.5 or 3.0 MU/m2 s.c. three times weekly). Unchanged capecitabine and its metabolites were analyzed in plasma using liquid chroatography/mass spectrometry in ten patients. RESULTS: The toxicity of combined capecitabine and rHuIFNalpha2a was moderate. Patients experienced mild nausea/vomiting (70%) and diarrhea (63%). The hand-foot syndrome was seen in 67% of patients and was generally mild, as was hematologic toxicity. Dose-limiting toxicity included diarrhea, mucositis, neutropenia and the hand-foot syndrome. The dose level recommended for further trials included capecitabine 1000 mg/m2 twice daily and rHuIFNalpha2a 3.0 MU/m2 three times weekly. One patient had a partial response of a liver lesion (duration > 200 days). Pharmacokinetic parameters of capecitabine and its metabolites (5'-deoxy-5-fluorouridine, 5-fluorouracil and alpha-fluoro-beta-alanine) were similar to those reported by other authors. There was rapid conversion to 5'-deoxyuridine. The peak plasma concentrations of capecitabine occurred between 0.5 and 3.0 h. CONCLUSIONS: The combination of capecitabine and rHuIFNalpha2a was well tolerated. The recommended dose levels for phase II trials are: rHuIFNalpha2a 3.0 MU/m2 s.c. three times weekly and oral capecitabine 1000 mg/m2 twice daily for 2 weeks. No evidence of an effect of rHuIFNalpha2a on the pharmacokinetics of capecitabine or its metabolites was apparent. A phase II trial in untreated patients with metastatic RCC is planned.

A point mutation in interleukin-2 that alters ligand internalization.

In previous studies, we have identified an interleukin-2 (IL-2) analog containing a point mutation at position 51 (T51P) that expresses nearly wild-type bioactivity, yet has approximately 10-fold lower receptor binding affinity. Since ligand-dependent receptor internalization may be the rate-limiting step controlling the duration of IL-2 receptor signaling, a reduction in the receptor internalization rate could contribute to the observed response enhancement for this analog. To evaluate this possibility, we compared the internalization of IL-2 and T51P in three separate assays. While the internalization rate for IL-2 agreed with values determined by others, the internalization of T51P was markedly reduced. The receptor binding rate constants for this analog were only slightly different; thus, altered binding kinetics could not explain the decreased internalization rate. The effects of reduced internalization were also observable in bioassays, where T51P maintained T-cell proliferation for a longer period compared with IL-2. These results indicate that the T51P point mutation reduces the receptor internalization rate compared with IL-2 in a fashion that is independent of the dissociation rate. This analog may represent a new approach to the preparation of cytokine analogs with potentiated agonist and antagonist properties.

Structural analogs of interleukin-2: a point mutation that facilitates biological response.

Interleukin-2 (IL-2) is an immunoregulatory cytokine whose biological effects are mediated through interaction with specific receptors on the surface of target cells. Due to its presumed role in generating a normal immune response, IL-2 is being evaluated for the treatment of a variety of tumors, in addition to infectious diseases. During the study of the structure-activity relationships for IL-2 and its receptors, one analog in which threonines at positions 41 and 51 were replaced by prolines (T41/51P) was found to possess apparent signaling abnormalities. Bioassays and receptor binding assays with human peripheral blood lymphocytes revealed the EC50 and Kd values of this analog to be 200 pM and 5.9 nM, respectively. Although the EC50 is greater and the receptor affinity of T41/51P is much weaker than that of wild-type IL-2, receptor occupancy versus biological response comparisons indicated that a much lower receptor occupancy was required to generate an equivalent biological response. Competitive receptor binding analyses with both intermediate affinity (beta/gamma subunit complex) and low affinity (alpha subunit) receptors were carried out to assess the origin of this phenomenon. Similar analyses of the singly substituted T41P and T51P analogs were carried out. From these studies, it was apparent that facilitated signaling was mainly attributable to position 51, whereas mutations at position 41 primarily influenced low affinity binding. The observation that the T51P analog facilitates response, compared with wild-type IL-2, may indicate a signaling-dependent conformational change in IL-2 upon receptor binding.

Mutagenic analysis of a receptor contact site on interleukin-2: preparation of an IL-2 analog with increased potency.

Interleukin-2 (IL-2) is a 133 amino acid alpha-helical protein secreted by activated T-cells. Combinatorial cassette mutagenesis was used to investigate the functional role of a continuous five amino acid region of IL-2 suspected to interact with the intermediate-affinity IL-2 receptor. A limited random library of IL-2 mutants was constructed in which residues 17-21 (Leu-Leu-Leu-Asp-Leu) were simultaneously mutated. The proteins were produced in an Escherichia coli expression system and screened in a biological assay for their ability to mediate the proliferation of a murine IL-2-dependent cell line. From the over 2600 clones examined, only 42 exhibited significant activity, confirming the functional importance of this region. Selected clones were purified and further characterized by biological and receptor binding assays. Viewed in the context of the recently revised 2.5-A crystal structure for IL-2, these results suggest the following conclusions: both Asp20 and Leu21, as shown by their sensitivity to mutation, are the functionally more important residues in this region, but for different reasons. Asp20 is solvent-accessible and likely plays a direct receptor contact role as previous studies have indicated. Leu21, in contrast, is completely buried in the hydrophobic core of the protein. Substitutions at this position, even a conservative Leu-->Val substitution, were found to perturb the precise hydrophobic packing arrangements that are critical for activity, resulting in a significant loss of function. In addition, one of the analogs identified in the screen was found to be 2-3 times more potent than the wild-type protein.