Parathyroid hormone-related protein protects against mammary tumor emergence and is associated with monocyte infiltration in ductal carcinoma in situ.

Parathyroid hormone-related protein (PTHrP) is required for mammary gland development and promotes the growth of breast cancer metastases within bone. However, there are conflicting reports of the prognostic significance of its expression in primary breast cancers. To study the role of PTHrP in early breast cancer, the effect of conditional deletion of PTHrP was examined in the context of neu-induced mammary tumorigenesis. Loss of PTHrP resulted in a higher tumor incidence. Transcriptional profiling of the tumors revealed that PTHrP influenced genes relevant to heterotypic cell signaling, including regulators of monocyte recruitment. Immunohistochemical analysis of human breast cancers revealed that PTHrP expression was associated with both HER-2/neu expression and macrophage infiltration in preinvasive ductal carcinoma in situ. The gene expression signature associated with loss of PTHrP expression in vivo correlated with poorer outcome in human breast cancer. Together, these data indicate that loss of PTHrP accelerates mammary tumorigenesis possibly by a non-cell-autonomous tumor suppressor pathway.

Effects of uteroplacental insufficiency and reducing litter size on maternal mammary function and postnatal offspring growth.

Human intrauterine growth restriction is often associated with uteroplacental insufficiency and a decline in nutrient and oxygen supply to the fetus. This study investigated the effects of uteroplacental insufficiency and intrauterine growth restriction (Restricted) or reducing litter size for normally grown pups (Reduced Litter) on maternal mammary development and function, milk composition, offspring milk intake, and their resultant effects on postnatal growth. Uteroplacental insufficiency was surgically induced by bilateral uterine vessel ligation on day 18 of gestation in the Wistar Kyoto rat. At birth, a group of sham control rats had their litter size reduced to five (Reduced Litter) to match that of the Restricted group. Cohorts of rats were terminally anesthetized on day 20 of gestation or day 6 of lactation, and a third group was studied throughout lactation. Restricted pups had a lower birth weight (by 16%) and litter size (by 36%) compared with controls, as well as reduced mammary parathyroid hormone-related protein content and milk ionic calcium concentrations associated with reduced total pup calcium. Restricted dams with lower circulating progesterone experienced premature lactogenesis, producing less milk per pup with altered composition compared with controls, further slowing growth during lactation. Reducing litter size of pups born of normal birth weight (Reduced Litter) was associated with decreased pup growth, highlighting the importance of appropriate controls. The present study demonstrates that uteroplacental insufficiency impairs mammary function, compromises milk quality and quantity, and reduces calcium transport into milk, further restraining postnatal growth.

Tumor-specific expression of alphavbeta3 integrin promotes spontaneous metastasis of breast cancer to bone.

INTRODUCTION: Studies in xenograft models and experimental models of metastasis have implicated several beta3 integrin-expressing cell populations, including endothelium, platelets and osteoclasts, in breast tumor progression. Since orthotopic human xenograft models of breast cancer are poorly metastatic to bone and experimental models bypass the formation of a primary tumor, however, the precise contribution of tumor-specific alphavbeta3 to the spontaneous metastasis of breast tumors from the mammary gland to bone remains unclear. METHODS: We used a syngeneic orthotopic model of spontaneous breast cancer metastasis to test whether exogenous expression of alphavbeta3 in a mammary carcinoma line (66cl4) that metastasizes to the lung, but not to bone, was sufficient to promote its spontaneous metastasis to bone from the mammary gland. The tumor burden in the spine and the lung following inoculation of alphavbeta3-expressing 66cl4 (66cl4beta3) tumor cells or control 66cl4pBabe into the mammary gland was analyzed by real-time quantitative PCR. The ability of these cells to grow and form osteolytic lesions in bone was determined by histology and tartrate-resistant acid phosphatase staining of bone sections following intratibial injection of tumor cells. The adhesive, migratory and invasive properties of 66cl4pBabe and 66cl4beta3 cells were evaluated in standard in vitro assays. RESULTS: The 66cl4beta3 tumors showed a 20-fold increase in metastatic burden in the spine compared with 66cl4pBabe. A similar trend in lung metastasis was observed. alphavbeta3 did not increase the proliferation of 66cl4 cells in vitro or in the mammary gland in vivo. Similarly, alphavbeta3 is not required for the proliferation of 66cl4 cells in bone as both 66cl4pBabe and 66cl4beta3 proliferated to the same extent when injected directly into the tibia. 66cl4beta3 tumor growth in the tibia, however, increased osteoclast recruitment and bone resorption compared with 66cl4 tumors. Moreover, alphavbeta3 increased 66cl4 tumor cell adhesion and alphavbeta3-dependent haptotactic migration towards bone matrix proteins, as well as their chemotactic response to bone-derived soluble factors in vitro. CONCLUSION: These results demonstrate for the first time that tumor-specific alphavbeta3 contributes to spontaneous metastasis of breast tumors to bone and suggest a critical role for this receptor in mediating chemotactic and haptotactic migration towards bone factors.

Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder.

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.

Genomic analysis of a spontaneous model of breast cancer metastasis to bone reveals a role for the extracellular matrix.

A clinically relevant model of spontaneous breast cancer metastasis to multiple sites, including bone, was characterized and used to identify genes involved in metastatic progression. The metastatic potential of several genetically related tumor lines was assayed using a novel real-time quantitative RT-PCR assay of tumor burden. Based on this assay, the tumor lines were categorized as nonmetastatic (67NR), weakly metastatic to lymph node (168FARN) or lung (66cl4), or highly metastatic to lymph node, lung, and bone (4T1.2 and 4T1.13). In vitro assays that mimic stages of metastasis showed that highly metastatic tumors lines were more adhesive, invasive, and migratory than the less metastatic lines. To identify metastasis-related genes in this model, each metastatic tumor was array profiled against the nonmetastatic 67NR using 15,000 mouse cDNA arrays. A significant proportion of genes relating to the extracellular matrix had elevated expression in highly metastatic tumors. The role of one of these genes, POEM, was further investigated in the model. In situ hybridization showed that POEM expression was specific to the tumor epithelium of highly metastatic tumors. Decreased POEM expression in 4T1.2 tumors significantly inhibited spontaneous metastasis to the lung, bone, and kidney. Taken together, our data support a role for the extracellular matrix in metastatic progression and describe, for the first time, a role for POEM in this process.

Uteroplacental restriction in the rat impairs fetal growth in association with alterations in placental growth factors including PTHrP.

During pregnancy, parathyroid hormone-related protein (PTHrP) is one of many growth factors that play important roles to promote fetal growth and development, including stimulation of placental calcium transport. Angiotensin II, acting through the AT(1a) receptor, is also known to promote placental growth. We examined the effects of bilateral uterine artery and vein ligation (restriction), which mimics placental insufficiency in humans, on growth, intrauterine PTHrP, placental AT(1a), and pup calcium. Growth restriction was surgically induced on day 18 of pregnancy in Wistar-Kyoto female rats by uterine vessel ligation. Uteroplacental insufficiency reduced fetal body weight by 15% and litter size (P < 0.001) compared with the control rats with no effect on placental weight or amniotic fluid volume. Uteroplacental insufficiency reduced placental PTHrP content by 46%, with increases in PTHrP (by 2.6-fold), parathyroid hormone (PTH)/PTHrP receptor (by 11.6-fold), and AT(1a) (by 1.7-fold) relative mRNA in placenta following restriction compared with results in control (P < 0.05). There were no alterations in uterine PTHrP and PTH/PTHrP receptor mRNA expression. Maternal and fetal plasma PTHrP and calcium concentrations were unchanged. Although fetal total body calcium was not altered, placental restriction altered perinatal calcium homeostasis, as evidenced by lower pup total body calcium after birth (P < 0.05). The increased uterine and amniotic fluid PTHrP (P < 0.05) may be an attempt to compensate for the induced impaired placental function. The present study demonstrates that uteroplacental insufficiency alters intrauterine PTHrP, placental AT(1a) expression, and perinatal calcium in association with a reduction in fetal growth. Uteroplacental insufficiency may provide an important model for exploring the early origins of adult diseases.

Co-expression of parathyroid hormone-related protein (PTHrP) and PTH/PTHrP receptor in cartilaginous tumours: a marker for malignancy?

AIM: Parathyroid hormone-related protein (PTHrP) is one of the critical factors for the differentiation and growth of chondrocytes. We examined the correlation between the co-expression of PTHrP and PTH/PTHrP receptor and the grade of malignancy in cartilaginous tumours. METHODS: We analysed PTHrP and PTH/PTHrP receptor expression in chondrosarcoma by immunohistochemistry and compared specific staining with the expression in benign cartilaginous tumours. There were 38 cartilaginous bone tumours consisting of 26 chondrosarcoma, six enchondroma and six osteochondroma. Chondrosarcoma were composed of 20 conventional chondrosarcoma (10 grade 1, seven grade 2, and three grade 3), two dedifferentiated chondrosarcoma, two clear cell chondrosarcoma, and two myxoid chondrosarcoma. We performed the standard peroxidase-labelled streptavidin-biotin detection method for immunohistochemistry using an antibody raised against PTHrP (1-14) and PTH/PTHrP receptor. The magnitude of receptor positivity of PTHrP and PTH/ PTHrP in each tumour was assessed as a percentage of PTHrP and PTH/PTHrP-positive cells per thousand tumour cells in the most histologically aggressive area of the tumour. RESULTS: All chondrosarcoma, five of six enchondroma, and four of six osteochondroma showed PTHrP-positive cells, and all chondrosarcoma, five of six enchondroma and five of six osteochondroma showed PTHrP receptor-positive cells. The grade of malignancy correlated with the percentage of both PTHrP and PTH/PTHrP receptor-positive tumour cells (P < 0.0001, either). Each grade of chondrosarcoma showed statistically higher expression of both PTHrP and PTH/PTHrP receptor than benign cartilaginous tumour. CONCLUSION: This is the first report of the co-expression of PTHrP and PTH/PTHrP receptor in chondrosarcoma. PTHrP and PTH/PTHrP receptor positivity may be valuable for differentiating between benign and malignant cartilaginous tumours.