Suppression of breast cancer growth and metastasis by a serpin myoepithelium-derived serine proteinase inhibitor expressed in the mammary myoepithelial cells

A serpin was identified in normal mammary gland by differential cDNA sequencing. In situ hybridization has detected this serpin exclusively in the myoepithelial cells on the normal and noninvasive mammary epithelial side of the basement membrane and thus was named myoepithelium-derived serine proteinase inhibitor (MEPI). No MEPI expression was detected in the malignant breast carcinomas. MEPI encodes a 405-aa precursor, including an 18-residue secretion signal with a calculated molecular mass of 46 kDa. The predicted sequence of the new protein shares 33% sequence identity and 58% sequence similarity to plasminogen activator inhibitor (PAI)-1 and PAI-2. To determine whether MEPI can modulate the in vivo growth and progression of human breast cancers, we transfected a full-length MEPI cDNA into human breast cancer cells and studied the orthotopic growth of MEPI-transfected vs. control clones in the mammary fat pad of athymic nude mice. Overexpression of MEPI inhibited the invasion of the cells in the in vitro invasion assay. When injected orthotopically into nude mice, the primary tumor volumes, axillary lymph node metastasis, and lung metastasis were significantly inhibited in MEPI-transfected clones as compared with controls. The expression of MEPI in myoepithelial cells may prevent breast cancer malignant progression leading to metastasis.

Calpain is a mediator of preservation-reperfusion injury in rat liver transplantation

Proteases as well as alterations in intracellular calcium have important roles in hepatic preservation-reperfusion injury, and increased calpain activity recently has been demonstrated in liver allografts. Experiments were designed to evaluate (i) hepatic cytosolic calpain activity during different periods of cold ischemia (CI), rewarming, or reperfusion, and (ii) effects of inhibition of calpain on liver graft function using the isolated perfused rat liver and arterialized orthotopic liver transplantation models. Calpain activity was assayed using the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin (AMC) and expressed as mean ± SD pmol AMC released/min per mg of cytosolic protein. Calpain activity rose significantly after 24 hr of CI in University of Wisconsin solution and further increased with longer preservation. Activity also increased within 30 min of rewarming, peaking at 120 min. Increased durations of CI preceding rewarming resulted in significantly higher activity (P < 0.01). Calpain activity increased rapidly upon reperfusion and was significantly enhanced by previous CI (P < 0.01). Calpain inhibition with Cbz-Val-Phe methyl ester significantly decreased aspartate aminotransferase released in the isolated perfused rat liver perfusate (P < 0.05). Duration of survival after orthotopic liver transplantation using livers cold-preserved for 40 hr was also significantly increased (P < 0.05) with calpain inhibitor. In conclusion, calpain proteases are activated during each phase of transplantation and are likely to play an important role in the mechanisms of preservation-reperfusion injury.

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.

Whole-body and intravital optical imaging of angiogenesis in orthotopically implanted tumors

The development of drugs for the control of tumor angiogenesis requires a simple, accurate, and economical assay for tumor-induced vascularization. We have adapted the orthotopic implantation model to angiogenesis measurement by using human tumors labeled with Aequorea victoria green fluorescent protein for grafting into nude mice. The nonluminous induced capillaries are clearly visible against the very bright tumor fluorescence examined either intravitally or by whole-body luminance in real time. The orthotopic implantation model of human cancer has been well characterized, and fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. Intravital images of orthotopically implanted human pancreatic tumors clearly show angiogenic capillaries at both primary and metastatic sites. A quantitative time course of angiogenesis was determined for an orthotopically growing human prostate tumor periodically imaged intravitally in a single nude mouse over a 19-day period. Whole-body optical imaging of tumor angiogenesis was demonstrated by injecting fluorescent Lewis lung carcinoma cells into the s.c. site of the footpad of nude mice. The footpad is relatively transparent, with comparatively few resident blood vessels, allowing quantitative imaging of tumor angiogenesis in the intact animal. Capillary density increased linearly over a 10-day period as determined by whole-body imaging. Similarly, the green fluorescent protein-expressing human breast tumor MDA-MB-435 was orthotopically transplanted to the mouse fat pad, where whole-body optical imaging showed that blood vessel density increased linearly over a 20-week period. These powerful and clinically relevant angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumor angiogenesis in physiological microenvironments.

Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts

Prostate stem-cell antigen (PSCA) is a cell-surface antigen expressed in normal prostate and overexpressed in prostate cancer tissues. PSCA expression is detected in over 80% of patients with local disease, and elevated levels of PSCA are correlated with increased tumor stage, grade, and androgen independence, including high expression in bone metastases. We evaluated the therapeutic efficacy of anti-PSCA mAbs in human prostate cancer xenograft mouse models by using the androgen-dependent LAPC-9 xenograft and the androgen-independent recombinant cell line PC3-PSCA. Two different anti-PSCA mAbs, 1G8 (IgG1κ) and 3C5 (IgG2aκ), inhibited formation of s.c. and orthotopic xenograft tumors in a dose-dependent manner. Furthermore, administration of anti-PSCA mAbs led to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies suggest PSCA as an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-PSCA mAbs for the treatment of local and metastatic prostate cancer.

PTEN controls tumor-induced angiogenesis

Mutations of the tumor suppressor PTEN, a phosphatase with specificity for 3-phosphorylated inositol phospholipids, accompany progression of brain tumors from benign to the most malignant forms. Tumor progression, particularly in aggressive and malignant tumors, is associated with the induction of angiogenesis, a process termed the angiogenic switch. Therefore, we tested whether PTEN regulates tumor progression by modulating angiogenesis. U87MG glioma cells stably reconstituted with PTEN cDNA were tested for growth in a nude mouse orthotopic brain tumor model. We observed that the reconstitution of wild-type PTEN had no effect on in vitro proliferation but dramatically decreased tumor growth in vivo and prolonged survival in mice implanted intracranially with these tumor cells. PTEN reconstitution diminished phosphorylation of AKT within the PTEN-reconstituted tumor, induced thrombospondin 1 expression, and suppressed angiogenic activity. These effects were not observed in tumors reconstituted with a lipid phosphatase inactive G129E mutant of PTEN, a result that provides evidence that the lipid phosphatase activity of PTEN regulates the angiogenic response in vivo. These data provide evidence that PTEN regulates tumor-induced angiogenesis and the progression of gliomas to a malignant phenotype via the regulation of phosphoinositide-dependent signals.

Liver colonization competence governs colon cancer metastasis.

Tumors that metastasize do so to preferred target organs. To explain this apparent specificity, Paget, > 100 years ago, formulated his seed and soil hypothesis; i.e., the cells from a given tumor would "seed'' only favorable "soil'' offered by certain groups. The hypothesis implies that cancer cells must find a suitable "soil'' in a target organ--i.e., one that supports colonization--for metastasis to occur. We demonstrate in this report that ability of human colon cancer cells to colonize liver tissue governs whether a particular colon cancer is metastatic. In the model used in this study, human colon tumors are transplanted into the nude mouse colon as intact tissue blocks by surgical orthotopic implantation. These implanted tumors closely simulate the metastatic behavior of the original human patient tumor and are clearly metastatic or nonmetastatic to the liver. Both classes of tumors were equally invasive locally into tissues and blood vessels. However, the cells from each class of tumor behave very differently when directly injected into nude mouse livers. Only cells from metastasizing tumors are competent to colonize after direct intrahepatic injection. Also, tissue blocks from metastatic tumors af fixed directly to the liver resulted in colonization, whereas no colonization resulted from nonmetastatic tumor tissue blocks even though some growth occurred within the tissue block itself. Thus, local invasion (injection) and even adhesion to the metastatic target organ (blocks) are not sufficient for metastasis. The results suggest that the ability to colonize the liver is the governing step in the metastasis of human colon cancer.