Mechanism of action of the third generation benzopyrans and evaluation of their broad anti-cancer activity in vitro and in vivo.

Successive rounds of chemical modification in three generations of benzopyran molecules have shown to select for different mechanisms of actions and progressive increases in anti-cancer activity. In this study, we investigated the mechanism of action of the third-generation benzopyran compounds, TRX-E-002-1 and TRX-E-009-1. High-content screening of a panel of 240 cancer cell lines treated with TRX-E-009-1 demonstrated it has broad anti-cancer potential. Within this screen, melanoma cell lines showed a range of sensitivities and subsequently a second independent panel of 21 melanoma 3D spheroid lines were assessed for their responses to both TRX-E-002-1 and TRX-E-009-1 compounds. Time-lapse microscopy illustrated both of these compounds caused mitotic delays in treated cells, resulting in either mitotic slippage or apoptosis. This finding along with immunostaining, in vitro polymerization assays, and animal experiments in both athymic and immunocompetent mice, demonstrates that these third-generation benzopyran compounds are potent tubulin polymerization inhibitors in vitro and in vivo, and this is the molecular basis of their anti-cancer activity in melanoma. These findings indicate these BP compounds may offer a novel anti-microtubule strategy for cancer intervention and provides the basis for further investigation into biomarkers of clinical sensitivity.

Regulation of colorectal cancer cell epithelial to mesenchymal transition by the renin angiotensin system.

BACKGROUND AND AIM: Epithelial to mesenchymal transition (EMT) is implicated in tumor progression. We aimed to determine if the renin angiotensin system has a role in colorectal cancer (CRC) cell EMT. METHODS: Human CRC cell lines DLD-1 and LIM2405 were used in wound scratch migration assays where they were treated with renin angiotensin system peptide ANG II alone or with blockers of ANG II type 1 or 2 receptors (AT1R and AT2R). Levels of epithelial (E-cadherin), mesenchymal (ZEB1, Vimentin) markers, inducible nitric oxide synthase (iNOS), and MMP9 were determined by flow cytometry. Mice bearing CRC liver metastases and treated with blockers for AT1R or AT2R were examined for ZEB1 and iNOS by immunohistochemistry. RESULTS: ANG II increased in-vitro CRC cell migration in both cell lines, this was inhibited by AT1R (IRB) or AT2R blockade (PD123319). DLD-1 cells treated with AT1R blocker resulted in increased E-cadherin, reduced ZEB1, and Vimentin expression compared with ANG II-treated cells. Treatment with AT2R blocker decreased E-cadherin, no change in ZEB1 or Vimentin expression. AT1R blockade increased iNOS and decreased MMP9 expression in DLD-1 and LIM2405 cells. AT2R blockade decreased iNOS and MMP9 expression in both cell lines. In vivo, ZEB1 staining was higher in ANG II-treated animals compared with control and AT1R blockade treated animals, while activation of the AT2R led to an increase in iNOS compared with control and AT1R blockade. CONCLUSIONS: ANG II-induced migration of CRC cells via both AT1 and AT2 receptors; the AT1R-mediated effects were associated with changes typical of EMT.

Blockade of MMP14 activity in murine breast carcinomas: implications for macrophages, vessels, and radiotherapy.

BACKGROUND: Matrix metalloproteinase (MMP) 14 may mediate tumor progression through vascular and immune-modulatory effects. METHODS: Orthotopic murine breast tumors (4T1 and E0771 with high and low MMP14 expression, respectively; n = 5-10 per group) were treated with an anti-MMP14 inhibitory antibody (DX-2400), IgG control, fractionated radiation therapy, or their combination. We assessed primary tumor growth, transforming growth factor ß (TGFß) and inducible nitric oxide synthase (iNOS) expression, macrophage phenotype, and vascular parameters. A linear mixed model with repeated observations, with Mann-Whitney or analysis of variance with Bonferroni post hoc adjustment, was used to determine statistical significance. All statistical tests were two-sided. RESULTS: DX-2400 inhibited tumor growth compared with IgG control treatment, increased macrophage numbers, and shifted the macrophage phenotype towards antitumor M1-like. These effects were associated with a reduction in active TGFß and SMAD2/3 signaling. DX-2400 also transiently increased iNOS expression and tumor perfusion, reduced tissue hypoxia (median % area: control, 20.2%, interquartile range (IQR) = 6.4%-38.9%; DX-2400: 1.2%, IQR = 0.2%-3.2%, P = .044), and synergistically enhanced radiation therapy (days to grow to 800mm(3): control, 12 days, IQR = 9-13 days; DX-2400 plus radiation, 29 days, IQR = 26-30 days, P < .001) in the 4T1 model. The selective iNOS inhibitor, 1400W, abolished the effects of DX-2400 on vessel perfusion and radiotherapy. On the other hand, DX-2400 was not capable of inducing iNOS expression or synergizing with radiation in E0771 tumors. CONCLUSION: MMP14 blockade decreased immunosuppressive TGFß, polarized macrophages to an antitumor phenotype, increased iNOS, and improved tumor perfusion, resulting in reduced primary tumor growth and enhanced response to radiation therapy, especially in high MMP14-expressing tumors.

Effects of vascular-endothelial protein tyrosine phosphatase inhibition on breast cancer vasculature and metastatic progression.

BACKGROUND: The solid tumor microvasculature is characterized by structural and functional abnormality and mediates several deleterious aspects of tumor behavior. Here we determine the role of vascular endothelial protein tyrosine phosphatase (VE-PTP), which deactivates endothelial cell (EC) Tie-2 receptor tyrosine kinase, thereby impairing maturation of tumor vessels. METHODS: AKB-9778 is a first-in-class VE-PTP inhibitor. We examined its effects on ECs in vitro and on embryonic angiogenesis in vivo using zebrafish assays. We studied the impact of AKB-9778 therapy on the tumor vasculature, tumor growth, and metastatic progression using orthotopic models of murine mammary carcinoma as well as spontaneous and experimental metastasis models. Finally, we used endothelial nitric oxide synthase (eNOS)-deficient mice to establish the role of eNOS in mediating the effects of VE-PTP inhibition. All statistical tests were two-sided. RESULTS: AKB-9778 induced ligand-independent Tie-2 activation in ECs and impaired embryonic zebrafish angiogenesis. AKB-9778 delayed the early phase of mammary tumor growth by maintaining vascular maturity (P < .01, t test); slowed growth of micrometastases (P < .01, χ(2) test) by preventing extravasation of tumor cells (P < 0.01, Fisher exact test), resulting in a trend toward prolonged survival (27.0 vs 36.5 days; hazard ratio of death = 0.33, 95% confidence interval = 0.11 to 1.03; P = .05, Mantel-Cox test); and stabilized established primary tumor blood vessels, enhancing tumor perfusion (P = .03 for 4T1 tumor model and 0.05 for E0771 tumor model, by two-sided t tests) and, hence, radiation response (P < .01, analysis of variance; n = 7 mice per group). The effects of AKB-9778 on tumor vessels were mediated in part by endothelial nitric oxide synthase activation. CONCLUSIONS: Our results demonstrate that pharmacological VE-PTP inhibition can normalize the structure and function of tumor vessels through Tie-2 activation, which delays tumor growth, slows metastatic progression, and enhances response to concomitant cytotoxic treatments.

The renin angiotensin system regulates Kupffer cells in colorectal liver metastases.

Blockade of the renin angiotensin system (RAS) can inhibit tumor growth and this may be mediated via undefined immunomodulatory actions. This study investigated the effects of RAS blockade on liver macrophages (Kupffer cells; KCs) in an orthotopic murine model of colorectal cancer (CRC) liver metastases. Here we showed that pharmacological targeting of the RAS [ANG II (31.25 µg/kg/h i.p.), ANG-(1-7) (24 µg/kg/h i.p.) or the ACE inhibitor; captopril (750 mg/kg/d i.p.)] altered endogenous KC numbers in the tumor-bearing liver throughout metastatic growth. Captopril, and to a lesser extent ANG-(1-7), increased KC numbers in the liver but not tumor. KCs were found to express the key RAS components: ACE and AT1R. Treatment with captopril and ANG II increased the number of AT1R-expressing KCs, although total KC numbers were not affected by ANG II. Captopril (0.1 µM) also increased macrophage invasion in vitro. Additionally, captopril was administered with KC depletion before tumor induction (day 0) or at established metastatic growth (day 18) using gadolinium chloride (GdCl 3; 20 mg/kg). Livers were collected at day 21 and quantitative stereology used as a measure of tumor burden. Captopril reduced growth of CRC liver metastases. However, when captopril was combined with early KC depletion (day 0) tumor growth was significantly increased compared with captopril alone. In contrast, late KC depletion (day 18) failed to influence the anti-tumor effects of captopril. The result of these studies suggests that manipulation of the RAS can alter KC numbers and may subsequently influence progression of CRC liver metastases.

Bimodal role of Kupffer cells during colorectal cancer liver metastasis.

Kupffer cells (KCs) are resident liver macrophages that play a crucial role in liver homeostasis and in the pathogenesis of liver disease. Evidence suggests KCs have both stimulatory and inhibitory functions during tumor development but the extent of these functions remains to be defined. Using KC depletion studies in an orthotopic murine model of colorectal cancer (CRC) liver metastases we demonstrated the bimodal role of KCs in determining tumor growth. KC depletion with gadolinium chloride before tumor induction was associated with an increased tumor burden during the exponential growth phase. In contrast, KC depletion at the late stage of tumor growth (day 18) decreased liver tumor load compared with non-depleted animals. This suggests KCs exhibit an early inhibitory and a later stimulatory effect. These two opposing functions were associated with changes in iNOS and VEGF expression as well as T-cell infiltration. KC depletion at day 18 increased numbers of CD3 (+) T cells and iNOS-expressing infiltrating cells in the tumor, but decreased the number of VEGF-expressing infiltrating cells. These alterations may be responsible for the observed reduction in tumor burden following depletion of pro-tumor KCs at the late stage of metastatic growth. Taken together, our results indicate that the bimodal role of KC activity in liver tumors may provide the key to timing immunomodulatory intervention for the treatment of CRC liver metastases.

Blockade of the renin-angiotensin system improves the early stages of liver regeneration and liver function.

BACKGROUND: Partial hepatectomy is the preferred option for selected patients with colorectal cancer liver metastases (CRCLM). Sufficient liver regeneration (LR) is essential for a successful outcome in these patients. The blockade of the renin-angiotensin system (RAS) reduces the growth of several tumor types. The RAS also acts as a regulator of liver fibrosis and potentially LR. The angiotensin-converting enzyme (ACE) inhibitor, captopril, significantly inhibits the growth of CRCLM, but its effect on LR remains undefined. METHODS: After 70% of partial hepatectomy, mice were randomly assigned to control or captopril-treated groups. LR was measured by liver-to-body weight ratio on days 1, 2, 4, 6, and 8. Hepatocyte proliferation, apoptosis and cell size, hepatic stellate cell (HSC) count, and sinusoidal endothelial cell density were quantified. Matrix metalloproteinase 9 (MMP-9) protein levels, liver injury markers, and RAS messenger RNA levels were also determined. RESULTS: At day 2, captopril increased liver-to-body weight ratio (56.5 ± 1.7 captopril versus 49.3 ± 2.4 control, P = 0.027). This was associated with increased HSC count (65.4 ± 4.8 cells per 100,000 µm(2), 48.7 ± 2.3, P = 0.007) and MMP-9 levels (0.68 ± 0.12 AU, 0.12 ± 0.04, P = 0.014). The messenger RNA levels of angiotensin-converting enzyme (P = 0.045) and angiotensin 1 receptor (P = 0.039) were reduced by captopril at day 2. CONCLUSION: Captopril enhanced early LR. This effect was associated with increased HSC numbers and MMP-9 protein, whereas hepatocyte proliferation was lower than controls. Captopril may provide a beneficial treatment option for the management of patients with CRCLM.

Combined targeting of HER2 and VEGFR2 for effective treatment of HER2-amplified breast cancer brain metastases.

Brain metastases are a serious obstacle in the treatment of patients with human epidermal growth factor receptor-2 (HER2)-amplified breast cancer. Although extracranial disease is controlled with HER2 inhibitors in the majority of patients, brain metastases often develop. Because these brain metastases do not respond to therapy, they are frequently the reason for treatment failure. We developed a mouse model of HER2-amplified breast cancer brain metastasis using an orthotopic xenograft of BT474 cells. As seen in patients, the HER2 inhibitors trastuzumab and lapatinib controlled tumor progression in the breast but failed to contain tumor growth in the brain. We observed that the combination of a HER2 inhibitor with an anti-VEGF receptor-2 (VEGFR2) antibody significantly slows tumor growth in the brain, resulting in a striking survival benefit. This benefit appears largely due to an enhanced antiangiogenic effect: Combination therapy reduced both the total and functional microvascular density in the brain xenografts. In addition, the combination therapy led to a marked increase in necrosis of the brain lesions. Moreover, we observed even better antitumor activity after combining both trastuzumab and lapatinib with the anti-VEGFR2 antibody. This triple-drug combination prolonged the median overall survival fivefold compared with the control-treated group and twofold compared with either two-drug regimen. These findings support the clinical development of this three-drug regimen for the treatment of HER2-amplified breast cancer brain metastases.

Altered efficacy of AT1R-targeted treatment after spontaneous cancer cell-AT1R upregulation.

BACKGROUND: Targeting of the renin angiotensin system (RAS) reduces tumour growth in experimental models of cancer. We aimed to establish if combined targeting of the 'classical' and 'alternative' arms of the RAS could result in synergistic inhibition of colorectal cancer (CRC) liver metastases. METHODS: Immediately following induction of CRC liver metastases through intrasplenic injection of murine CRC cells, treatment with irbesartan (AT1R blocker; 50 mg/kg/day s.c.), captopril (ACE inhibitor; 750 mg/kg/day i.p.), CGP42112A (AT2R agonist; 0.6 µg/kg/hr i.p.), and/or ANG-(1-7) (24 µg/kg/hr i.p.) began and continued for 21 days. Liver to body weight ratio and/or stereology were used as a measure of tumour burden. Immunohistochemistry was used to determine AT1R and VEGF expression as well as proliferation (Ki67), apoptosis (active caspase 3) and angiogenesis (CD34). RESULTS: Combined RAS therapies failed to improve upon single arm therapies. However, while irbesartan previously inhibited tumour growth in this model, in the current experiments irbesartan failed to affect tumour burden. Subsequent analysis showed a cancer-cell specific upregulation of the angiotensin II type I receptor (AT1R) in irbesartan-insensitive compared to irbesartan-sensitive tumours. The upregulation of AT1R was associated with an increase in proliferation and VEGF expression by cancer cells. While animals bearing irbesartan-sensitive tumours showed a marked decrease in the number of proliferating cells in the liver and VEGF-expressing infiltrating cells in the tumour following AT1R treatment, these were unchanged by treatment in animals bearing irbesartan-insensitive (high AT1R expressing) tumours. CONCLUSIONS: Although the results do not support increased efficacy of combined treatment, they provide intriguing evidence of the importance of RAS expression in determining patient response and tumour growth potential and suggest that components of the RAS could be used as biomarkers to aid in patient selection.

Liver regeneration and tumour stimulation: implications of the renin-angiotensin system.

Liver resection is the most effective treatment for primary liver tumours and metastasis to the liver, and remains the only potentially long-term curative therapy for patients with colorectal cancer (CRC) liver metastases. Nevertheless, there is a significant incidence of tumour recurrence following liver resection. Cellular and molecular changes resulting from resection and the subsequent liver regeneration process may influence the kinetics of tumour growth, contributing to recurrence. Although commonly associated with the systemic homeostasis of blood pressure, fluid and electrolyte, the renin-angiotensin system (RAS) has recently been shown to play a role in regulating cell proliferation, apoptosis and angiogenesis in local organs as well as in malignancies. An electronic search of the English literature on the role of the RAS in liver regeneration and tumourigenesis was performed using PubMed, with additional relevant articles sourced from reference lists. Studies have shown that the blockade of the RAS pathway stimulates liver regeneration and inhibits tumour progression. An understanding of the role of RAS in liver regeneration and tumourigenesis may enable alternative strategies to improve patient outcome and survival after liver resection. This review will discuss the role of the RAS in liver regeneration and in tumour recurrence post-liver resection. The potential of the RAS as a novel therapeutic target for CRC liver metastases patients undergoing liver resection will be outlined.

Targeting the angiotensin II type 2 receptor (AT2R) in colorectal liver metastases.

BACKGROUND: Blockade of the angiotensin (ANG) II type 1 receptor (AT1R) inhibits tumour growth in several cancers, including colorectal cancer (CRC) liver metastases. While AT1R blockade has been extensively studied, the potential of targeting the antagonistically acting AT2R in cancer has not been investigated. This study examined the effect of AT2R activation with the agonist CGP42112A in a mouse model of CRC liver metastases. RESULTS: In vitro, mouse CRC cell (MoCR) proliferation was inhibited by treatment with CGP42112A in a dose dependent manner while apoptosis was increased. Immunofluorescent staining for key signalling and secondary messengers, PLA2 and iNOS, were also increased by CGP42112A treatment in vitro. Immunohistochemical staining for proliferation (PCNA) and the apoptosis (active caspase 3) markers confirmed a CGP42112A-associated inhibition of proliferation and induction of apoptosis of mouse CRC cells (MoCR) in vivo. However, angiogenesis and vascular endothelial growth factor (VEGF) appeared to be increased by CGP42112A treatment in vivo. This increase in VEGF secretion by MoCRs was confirmed in vitro. Despite this apparent pro-angiogenic effect, a syngenic orthotopic mouse model of CRC liver metastases showed a reduction in liver to body weight ratio, an indication of tumour burden, following CGP42112A treatment compared to untreated controls. CONCLUSIONS: These results suggest that AT2R activation might provide a novel target to inhibit tumour growth. Its potential to stimulate angiogenesis could be compensated by combination with anti-angiogenic agents.

Changes in the renin angiotensin system during the development of colorectal cancer liver metastases.

BACKGROUND: Blockade of the renin angiotensin system (RAS) via angiotensin I converting enzyme (ACE) inhibition reduces growth of colorectal cancer (CRC) liver metastases in a mouse model. In this work we defined the expression of the various components of the RAS in both tumor and liver during the progression of this disease. METHODS: Immunohistochemistry and quantitative RT-PCR was used to examine RAS expression in a mouse CRC liver metastases model. CRC metastases and liver tissue was assessed separately at key stages of CRC liver metastases development in untreated (control) mice and in mice treated with the ACE inhibitor captopril (750 mg/kg/day). Non-tumor induced (sham) mice indicated the effect of tumors on normal liver RAS. The statistical significance of multiple comparisons was determined using one-way analysis of variance followed by Bonferroni adjustment with SAS/STAT software. RESULTS: Reduced volume of CRC liver metastases with captopril treatment was evident. Local RAS of CRC metastases differed from the surrounding liver, with lower angiotensin II type 1 receptor (AT1R) expression but increased ANG-(1-7) receptor (MasR) compared to the liver. The AT1R localised to cancer and stromal infiltrating cells, while other RAS receptors were detected in cancer cells only. Tumor induction led to an initial increase in AT1R and ACE expression while captopril treatment significantly increased ACE expression in the final stages of tumor growth. Conversely, captopril treatment decreased expression of AT1R and angiotensinogen. CONCLUSIONS: These results demonstrate significant changes in RAS expression in the tumor-bearing captopril treated liver and in CRC metastases. The data suggests the existence of a tumor-specific RAS that can be independently targeted by RAS blockade.

Analysis of the platypus genome suggests a transposon origin for mammalian imprinting.

BACKGROUND: Genomic imprinting is an epigenetic phenomenon that results in monoallelic gene expression. Many hypotheses have been advanced to explain why genomic imprinting evolved in mammals, but few have examined how it arose. The host defence hypothesis suggests that imprinting evolved from existing mechanisms within the cell that act to silence foreign DNA elements that insert into the genome. However, the changes to the mammalian genome that accompanied the evolution of imprinting have been hard to define due to the absence of large scale genomic resources between all extant classes. The recent release of the platypus genome has provided the first opportunity to perform comparisons between prototherian (monotreme; which appear to lack imprinting) and therian (marsupial and eutherian; which have imprinting) mammals. RESULTS: We compared the distribution of repeat elements known to attract epigenetic silencing across the entire genome from monotremes and therian mammals, particularly focusing on the orthologous imprinted regions. There is a significant accumulation of certain repeat elements within imprinted regions of therian mammals compared to the platypus. CONCLUSIONS: Our analyses show that the platypus has significantly fewer repeats of certain classes in the regions of the genome that have become imprinted in therian mammals. The accumulation of repeats, especially long terminal repeats and DNA elements, in therian imprinted genes and gene clusters is coincident with, and may have been a potential driving force in, the development of mammalian genomic imprinting. These data provide strong support for the host defence hypothesis.

Genomic imprinting in marsupial placentation.

Genomic imprinting is a widespread epigenetic phenomenon in eutherian mammals, which regulates many aspects of growth and development. Parental conflict over the degree of maternal nutrient transfer is the favoured hypothesis for the evolution of imprinting. Marsupials, like eutherian mammals, are viviparous but deliver an altricial young after a short gestation supported by a fully functional placenta, so can shed light on the evolution and time of acquisition of genomic imprinting. All orthologues of eutherian imprinted genes examined have a conserved expression in the marsupial placenta regardless of their imprint status. Differentially methylated regions (DMRs) are the most common mechanism controlling genomic imprinting in eutherian mammals, but none were found in the marsupial imprinted orthologues of IGF2 receptor (IGF2R), INS or mesoderm-specific transcript (MEST). Instead, histone modification appears to be the mechanism used to silence these genes. At least three genes in marsupials have DMRs: H19, IGF2 and PEG10. PEG10 is particularly interesting as it is derived from a retrotransposon, providing the first direct evidence that retrotransposon insertion can drive the evolution of an imprinted region and of a DMR in mammals. The insertion occurred after the prototherian-therian mammal divergence, suggesting that there may have been strong selection for the retention of imprinted regions that arose during the evolution of placentation. There is currently no evidence for genomic imprinting in the egg-laying monotreme mammals. However, since these mammals do have a short-lived placenta, imprinting appears to be correlated with viviparity but not placentation.

The renin-angiotensin system and malignancy.

The renin-angiotensin system (RAS) is usually associated with its systemic action on cardiovascular homoeostasis. However, recent studies suggest that at a local tissue level, the RAS influences tumour growth. The potential of the RAS as a target for cancer treatment and the suggested underlying mechanisms of its paracrine effects are reviewed here. These include modulation of angiogenesis, cellular proliferation, immune responses and extracellular matrix formation. Knowledge of the RAS has increased dramatically in recent years with the discovery of new enzymes, peptides and feedback mechanisms. The local RAS appears to influence tumour growth and metastases and there is evidence of tissue- and tumour-specific differences. Recent experimental studies provide strong evidence that drugs that inhibit the RAS have the potential to reduce cancer risk or retard tumour growth and metastases. Manipulation of the RAS may, therefore, provide a safe and inexpensive anticancer strategy.

Evolution of the CDKN1C-KCNQ1 imprinted domain.

BACKGROUND: Genomic imprinting occurs in both marsupial and eutherian mammals. The CDKN1C and IGF2 genes are both imprinted and syntenic in the mouse and human, but in marsupials only IGF2 is imprinted. This study examines the evolution of features that, in eutherians, regulate CDKN1C imprinting. RESULTS: Despite the absence of imprinting, CDKN1C protein was present in the tammar wallaby placenta. Genomic analysis of the tammar region confirmed that CDKN1C is syntenic with IGF2. However, there are fewer LTR and DNA elements in the region and in intron 9 of KCNQ1. In addition there are fewer LINEs in the tammar compared with human and mouse. While the CpG island in intron 10 of KCNQ1 and promoter elements could not be detected, the antisense transcript KCNQ1OT1 that regulates CDKN1C imprinting in human and mouse is still expressed. CONCLUSION: CDKN1C has a conserved function, likely antagonistic to IGF2, in the mammalian placenta that preceded its acquisition of imprinting. CDKN1C resides in synteny with IGF2, demonstrating that imprinting of the two genes did not occur concurrently to balance maternal and paternal influences on the growth of the placenta. The expression of KCNQ1OT1 in the absence of CDKN1C imprinting suggests that antisense transcription at this locus preceded imprinting of this domain. These findings demonstrate the stepwise accumulation of control mechanisms within imprinted domains and show that CDKN1C imprinting cannot be due to its synteny with IGF2 or with its placental expression in mammals.

Expression and protein localisation of IGF2 in the marsupial placenta.

BACKGROUND: In eutherian mammals, genomic imprinting is critical for normal placentation and embryo survival. Insulin-like growth factor 2 (IGF2) is imprinted in the placenta of both eutherians and marsupials, but its function, or that of any imprinted gene, has not been investigated in any marsupial. This study examines the role of IGF2 in the yolk sac placenta of the tammar wallaby, Macropus eugenii. RESULTS: IGF2 mRNA and protein were produced in the marsupial placenta. Both IGF2 receptors were present in the placenta, and presumably mediate IGF2 mitogenic actions. IGF2 mRNA levels were highest in the vascular region of the yolk sac placenta. IGF2 increased vascular endothelial growth factor expression in placental explant cultures, suggesting that IGF2 promotes vascularisation of the yolk sac. CONCLUSION: This is the first demonstration of a physiological role for any imprinted gene in marsupial placentation. The conserved imprinting of IGF2 in this marsupial and in all eutherian species so far investigated, but not in monotremes, suggests that imprinting of this gene may have originated in the placenta of the therian ancestor.

Insulin is imprinted in the placenta of the marsupial, Macropus eugenii.

Therian mammals (marsupials and eutherians) rely on a placenta for embryo survival. All mammals have a yolk sac, but while both chorio-allantoic and chorio-vitelline (yolk sac) placentation can occur, most marsupials only develop a yolk sac placenta. Insulin (INS) is unusual in that it is the only gene that is imprinted exclusively in the yolk sac placenta. Marsupials, therefore, provide a unique opportunity to examine the conservation of INS imprinting in mammalian yolk sac placentation. Marsupial INS was cloned and its imprint status in the yolk sac placenta of the tammar wallaby, Macropus eugenii, examined. In two informative individuals of the eight that showed imprinting, INS was paternally expressed. INS protein was restricted to the yolk sac endoderm, while insulin receptor, IR, protein was additionally expressed in the trophoblast. INS protein increased during late gestation up to 2 days before birth, but was low the day before and on the day of birth. The conservation of imprinted expression of insulin in the yolk sac placenta of divergent mammalian species suggests that it is of critical importance in the yolk sac placenta. The restriction of imprinting to the yolk sac suggests that imprinting of INS evolved in the chorio-vitelline placenta independently of other tissues in the therian ancestor of marsupials and eutherians.

Recent assembly of an imprinted domain from non-imprinted components.

Genomic imprinting, representing parent-specific expression of alleles at a locus, raises many questions about how--and especially why--epigenetic silencing of mammalian genes evolved. We present the first in-depth study of how a human imprinted domain evolved, analyzing a domain containing several imprinted genes that are involved in human disease. Using comparisons of orthologous genes in humans, marsupials, and the platypus, we discovered that the Prader-Willi/Angelman syndrome region on human Chromosome 15q was assembled only recently (105-180 million years ago). This imprinted domain arose after a region bearing UBE3A (Angelman syndrome) fused with an unlinked region bearing SNRPN (Prader-Willi syndrome), which had duplicated from the non-imprinted SNRPB/B'. This region independently acquired several retroposed gene copies and arrays of small nucleolar RNAs from different parts of the genome. In their original configurations, SNRPN and UBE3A are expressed from both alleles, implying that acquisition of imprinting occurred after their rearrangement and required the evolution of a control locus. Thus, the evolution of imprinting in viviparous mammals is ongoing.