GT198 Expression Defines Mutant Tumor Stroma in Human Breast Cancer.

Human breast cancer precursor cells remain to be elucidated. Using breast cancer gene product GT198 (PSMC3IP; alias TBPIP or Hop2) as a unique marker, we revealed the cellular identities of GT198 mutant cells in human breast tumor stroma. GT198 is a steroid hormone receptor coactivator and a crucial factor in DNA repair. Germline mutations in GT198 are present in breast and ovarian cancer families. Somatic mutations in GT198 are present in ovarian tumor stromal cells. Herein, we show that human breast tumor stromal cells carry GT198 somatic mutations and express cytoplasmic GT198 protein. GT198(+) stromal cells share vascular smooth muscle cell origin, including myoepithelial cells, adipocytes, capillary pericytes, and stromal fibroblasts. Frequent GT198 mutations are associated with GT198(+) tumor stroma but not with GT198(-) tumor cells. GT198(+) progenitor cells are mostly capillary pericytes. When tested in cultured cells, mutant GT198 induces vascular endothelial growth factor promoter, and potentially promotes angiogenesis and adipogenesis. Our results suggest that multiple lineages of breast tumor stromal cells are mutated in GT198. These findings imply the presence of mutant progenitors, whereas their descendants, carrying the same GT198 mutations, are collectively responsible for forming breast tumor microenvironment. GT198 expression is, therefore, a specific marker of mutant breast tumor stroma and has the potential to facilitate diagnosis and targeted treatment of human breast cancer.

Human ovarian cancer stroma contains luteinized theca cells harboring tumor suppressor gene GT198 mutations.

Ovarian cancer is a highly lethal gynecological cancer, and its causes remain to be understood. Using a recently identified tumor suppressor gene, GT198 (PSMC3IP), as a unique marker, we searched for the identity of GT198 mutant cells in ovarian cancer. GT198 has germ line mutations in familial and early onset breast and ovarian cancers and recurrent somatic mutations in sporadic fallopian tube cancers. GT198 protein has been shown as a steroid hormone receptor coregulator and also as a crucial factor in DNA repair. In this study, using GT198 as a marker for microdissection, we find that ovarian tumor stromal cells harboring GT198 mutations are present in various types of ovarian cancer including high and low grade serous, endometrioid, mucinous, clear cell, and granulosa cell carcinomas and in precursor lesions such as inclusion cysts. The mutant stromal cells consist of a luteinized theca cell lineage at various differentiation stages including CD133(+), CD44(+), and CD34(+) cells, although the vast majority of them are differentiated overexpressing steroidogenic enzyme CYP17, a theca cell-specific marker. In addition, wild type GT198 suppresses whereas mutant GT198 protein stimulates CYP17 expression. The chromatin-bound GT198 on the human CYP17 promoter is decreased by overexpressing mutant GT198 protein, implicating the loss of wild type suppression in mutant cells. Together, our results suggest that GT198 mutant luteinized theca cells overexpressing CYP17 are common in ovarian cancer stroma. Because first hit cancer gene mutations would specifically mark cancer-inducing cells, the identification of mutant luteinized theca cells may add crucial evidence in understanding the cause of human ovarian cancer.

Decoding cancer and herbal renaissance.

The original notion in quest of cancer targets to end cancer still stands, yet the secret of common human cancer was concealed by a chicken-egg paradox. Solid tumors initiate in the tumor microenvironment from rare stem cells, which express a mutant target protein as their specific marker. For decades, the stem cell and target protein cannot paradoxically be found one without first finding the other. With combined evidence from genetics, pathology, stem cell biology, clinical oncology, and herbal medicine in particular, this paradox is resolved. Historical successful anticancer herbs, together with clinical oncology drugs, paved the way to decode cancer. In solid tumors, the liable stem cells are pericyte stem cells on blood vessels in the tumor microenvironment inducing angiogenesis. One identified target protein in pericytes is a DNA repair factor and transcriptional regulator named GT198 (gene symbol PSMC3IP, alias name Hop2). Since GT198 is found as a direct drug target of many chemotherapy drugs and clinically successful anticancer herbs, more herbal medicines worldwide can now be screened against this target. In the near future, safer and more effective natural herbal medicines could systematically treat common solid tumors. This review discusses a unified theory of cancer and diseases in which pericyte stem cells are fundamental to both. It also reveals a new approach to identifying multi-functional herbs. Unlocking herbal targets in stem cells enables effective herbal identification and, in turn, awakens the herbal renaissance.

GT198 Is a Target of Oncology Drugs and Anticancer Herbs.

Tumor angiogenesis is a hallmark of cancer. Therapeutic drug inhibitors targeting angiogenesis are clinically effective. We have previously identified GT198 (gene symbol PSMC3IP, also known as Hop2) as an oncoprotein that induces tumor angiogenesis in human cancers, including oral cancer. In this study, we show that the GT198 protein is a direct drug target of more than a dozen oncology drugs and several clinically successful anticancer herbs. GT198 is a DNA repair protein that binds to DNA. Using an in vitro DNA-binding assay, we tested the approved oncology drug set VII from the National Cancer Institute containing 129 oncology drugs. Identified GT198 inhibitors include but are not limited to mitoxantrone, doxorubicin, paclitaxel, etoposide, dactinomycin, and imatinib. Paclitaxel and etoposide have higher binding affinities, whereas doxorubicin has higher binding efficacy due to competitive inhibition. GT198 shares protein sequence homology with DNA topoisomerases, which are known drug targets, so that GT198 is likely a new drug target previously unrecognized. To seek more powerful GT198 inhibitors, we further tested several anticancer herbal extracts. The positive anticancer herbs with high affinity and high efficacy are all clinically successful ones, including allspice from Jamaica, Gleditsia sinensis or honey locust from China, and BIRM from Ecuador. Partial purification of allspice using an organic chemical approach demonstrated great feasibility of natural product purification, when the activity is monitored by the in vitro DNA-binding assay using GT198 as a target. Together, our study reveals GT198 as a new targeting mechanism for existing oncology drugs. The study also delivers an excellent drug target suitable for compound identification and natural product purification. In particular, this study opens an opportunity to rapidly identify drugs with high efficacy and low toxicity from nature.

Functional pre- mRNA trans-splicing of coactivator CoAA and corepressor RBM4 during stem/progenitor cell differentiation.

Alternative splicing yields functionally distinctive gene products, and their balance plays critical roles in cell differentiation and development. We have previously shown that tumor-associated enhancer loss in coactivator gene CoAA leads to its altered alternative splicing. Here we identified two intergenic splicing variants, a zinc finger-containing coactivator CoAZ and a non-coding transcript ncCoAZ, between CoAA and its downstream corepressor gene RBM4. During stem/progenitor cell neural differentiation, we found that the switched alternative splicing and trans-splicing between CoAA and RBM4 transcripts result in lineage-specific expression of wild type CoAA, RBM4, and their variants. Stable expression of CoAA, RBM4, or their variants prevents the switch and disrupts the embryoid body formation. In addition, CoAA and RBM4 counter-regulate the target gene Tau at exon 10, and their splicing activities are subjected to the control by each splice variant. Further phylogenetic analysis showed that mammalian CoAA and RBM4 genes share common ancestry with the Drosophila melanogaster gene Lark, which is known to regulate early development and circadian rhythms. Thus, the trans-splicing between CoAA and RBM4 transcripts may represent a required regulation preserved during evolution. Our results demonstrate that a linked splicing control of transcriptional coactivator and corepressor is involved in stem/progenitor cell differentiation. The alternative splicing imbalance of CoAA and RBM4, because of loss of their common enhancer in cancer, may deregulate stem/progenitor cell differentiation.

Formation and Characterization of Zein-Based Oleogels.

Oleogels are interesting as a result of their ability to hold large amounts of oil in a semi-solid gel structure. In the food industry, oleogels are most often investigated as substitutes for saturated and trans fats. In this work, the lyotropic formation of ethanol/zein/oleic acid gels was observed qualitatively and ternary phase diagrams were constructed to map the observations. The viscoelastic parameters G' and G'' were measured to confirm gel formation as observed in phase diagrams. Ultrasmall X-ray scattering was used to study the microstructural organization of ethanol/zein/oleic acid gels. Data suggested that the primary unit or building block for gel network structures was the rod-shaped zein molecule. Ultrasmall X-ray data suggested that zein/oleic acid gels have a highly organized microstructure, possibly the result of zein self-assembly. Zein was considered an effective oleogelator in ethanol/zein/oleic acid systems.

Oncoprotein GT198 vaccination delays tumor growth in MMTV-PyMT mice.

Targeting early lesion in breast cancer is more therapeutically effective. We have previously identified an oncoprotein GT198 (PSMC3IP) in human breast cancer. Here we investigated GT198 in MMTV-PyMT mouse mammary gland tumors and found that GT198 is a shared early lesion in both species. Similar to human breast cancer even before a tumor appears, cytoplasmic GT198 is overexpressed in mouse tumor stroma including pericyte stem cells, descendent adipocytes, fibroblasts, and myoepithelial cells. Using recombinant GT198 protein as an antigen, we vaccinated MMTV-PyMT mice and found that the GT198 vaccine delayed mouse tumor growth and reduced lung metastasis. The antitumor effects were linearly correlated with vaccinated mouse serum titers of GT198 antibody, which recognized cell surface GT198 protein on viable tumor cells confirmed by FACS. Furthermore, GT198+ tumor cells isolated from MMTV-PyMT tumor induced faster tumor growths than GT198- cells when re-implanted into normal FVB/N mice. Together, this first study of GT198 vaccine in mouse showed its effectiveness in antitumor and anti-metastasis. The finding supports GT198 as a potential target in human immunotherapy since GT198 defect is shared in both human and mouse.

Switched alternative splicing of oncogene CoAA during embryonal carcinoma stem cell differentiation.

Alternative splicing produces functionally distinct proteins participating in cellular processes including differentiation and development. CoAA is a coactivator that regulates transcription-coupled splicing and its own pre-mRNA transcript is alternatively spliced. We show here that the CoAA gene is embryonically expressed and alternatively spliced in multiple tissues to three splice variants, CoAA, CoAM and CoAR. During retinoic-acid-induced P19 stem cell differentiation, the expression of CoAA undergoes a rapid switch to its dominant negative splice variant CoAM in the cavity of the embryoid body. CoAM functionally inhibits CoAA, and their switched expression up-regulates differentiation marker Sox6. Using a CoAA minigene cassette, we find that the switched alternative splicing of CoAA and CoAM is regulated by the cis-regulating sequence upstream of the CoAA basal promoter. Consistent to this, we show that p54(nrb) and PSF induce CoAM splice variant through the cis-regulating sequence. We have previously shown that the CoAA gene is amplified in human cancers with a recurrent loss of this cis-regulating sequence. These results together suggest that the upstream regulatory sequence contributes to alternative splicing of the CoAA gene during stem cell differentiation, and its selective loss in human cancers potentially deregulates CoAA alternative splicing and alters stem cell differentiation.

Identification and characterization of a tissue-specific coactivator, GT198, that interacts with the DNA-binding domains of nuclear receptors.

Gene activation mediated by nuclear receptors is regulated in a tissue-specific manner and requires interactions between nuclear receptors and their cofactors. Here, we identified and characterized a tissue-specific coactivator, GT198, that interacts with the DNA-binding domains of nuclear receptors. GT198 was originally described as a genomic transcript that mapped to the human breast cancer susceptibility locus 17q12-q21 with unknown function. We show that GT198 exhibits a tissue-specific expression pattern in which its mRNA is elevated in testis, spleen, thymus, pituitary cells, and several cancer cell lines. GT198 is a 217-amino-acid nuclear protein that contains a leucine zipper required for its dimerization. In vitro binding and yeast two-hybrid assays indicated that GT198 interacted with nuclear receptors through their DNA-binding domains. GT198 potently stimulated transcription mediated by estrogen receptor alpha and beta, thyroid hormone receptor beta1, androgen receptor, glucocorticoid receptor, and progesterone receptor. However, the action of GT198 was distinguishable from that of the ligand-binding domain-interacting nuclear receptor coactivators, such as TRBP, CBP, and SRC-1, with respect to basal activation and hormone sensitivity. Furthermore, protein kinase A, protein kinase C, and mitogen-activated protein kinase can phosphorylate GT198 in vitro, and cotransfection of these kinases regulated the transcriptional activity of GT198. These data suggest that GT198 is a tissue-specific, kinase-regulated nuclear receptor coactivator that interacts with the DNA-binding domains of nuclear receptors.

CoAA, a nuclear receptor coactivator protein at the interface of transcriptional coactivation and RNA splicing.

We have shown that steroid hormones coordinately control gene transcriptional activity and splicing decisions in a promoter-dependent manner. Our hypothesis is that a subset of hormonally recruited coregulators involved in regulation of promoter transcriptional activity also directly participate in alternative RNA splicing decisions. To gain insight into the molecular mechanisms by which transcriptional coregulators could control splicing decisions, we focused our attention on a recently identified coactivator, CoAA. This heterogeneous nuclear ribonucleoprotein (hnRNP)-like protein interacts with the transcriptional coregulator TRBP, a protein recruited to target promoters through interactions with activated nuclear receptors. Using transcriptional and splicing reporter genes driven by different promoters, we observed that CoAA mediates transcriptional and splicing effects in a promoter-preferential manner. We compared the activity of CoAA to the activity of other hnRNP-related proteins that, like CoAA, contain two N-terminal RNA recognition motifs (RRMs) followed by a C-terminal auxiliary domain and either have or have not been implicated in transcriptional control. By swapping either CoAA RRMs or the CoAA auxiliary domain with the corresponding domains of the proteins selected, we showed that depending on the promoter, the RRMs and the auxiliary domain of CoAA are differentially engaged in transcription. This contributes to the promoter-preferential effects mediated by CoAA on RNA splicing during the course of steroid hormone action.

Malignant pericytes expressing GT198 give rise to tumor cells through angiogenesis.

Angiogenesis promotes tumor development. Understanding the crucial factors regulating tumor angiogenesis may reveal new therapeutic targets. Human GT198 (PSMC3IP or Hop2) is an oncoprotein encoded by a DNA repair gene that is overexpressed in tumor stromal vasculature to stimulate the expression of angiogenic factors. Here we show that pericytes expressing GT198 give rise to tumor cells through angiogenesis. GT198+ pericytes and perivascular cells are commonly present in the stromal compartment of various human solid tumors and rodent xenograft tumor models. In human oral cancer, GT198+ pericytes proliferate into GT198+ tumor cells, which migrate into lymph nodes. Increased GT198 expression is associated with increased lymph node metastasis and decreased progression-free survival in oral cancer patients. In rat brain U-251 glioblastoma xenografts, GT198+ pericytes of human tumor origin encase endothelial cells of rat origin to form mosaic angiogenic blood vessels, and differentiate into pericyte-derived tumor cells. The net effect is continued production of glioblastoma tumor cells from malignant pericytes via angiogenesis. In addition, activation of GT198 induces the expression of VEGF and promotes tube formation in cultured U251 cells. Furthermore, vaccination using GT198 protein as an antigen in mouse xenograft of GL261 glioma delayed tumor growth and prolonged mouse survival. Together, these findings suggest that GT198-expressing malignant pericytes can give rise to tumor cells through angiogenesis, and serve as a potential source of cells for distant metastasis. Hence, the oncoprotein GT198 has the potential to be a new target in anti-angiogenic therapies in human cancer.

Ser-884 adjacent to the LXXLL motif of coactivator TRBP defines selectivity for ERs and TRs.

Ligand-dependent interaction of nuclear receptors and coactivators is a critical step in nuclear receptor-mediated transcriptional regulation. TR-binding protein (TRBP) interacts with nuclear receptors through a single LXXLL motif. Evidence suggested that the sequences flanking the LXXLL motif in a number of coactivators determine receptor selectivity. We performed mutagenesis studies at residues adjacent to the TRBP LXXLL motif and identified S884 of TRBP at the -3 position of the LXXLL motif as a key residue for receptor selectivity. Analysis of in vitro and in vivo receptor interactions with TRBP suggested that S884 allowed selective interactions for ERbeta, TR, and RXR vs. ERalpha. Transient transfection studies further confirmed that the LXXLL-binding affinity correlates with TRBP transcriptional activity. Consistent with the structural modeling, an E380G substitution within ERalpha altered the binding to TRBP mutants, demonstrating the direct contact between TRBP S884 and ERalpha E380, which is a residue that distinguishes receptor subclasses. Furthermore, S884 can be phosphorylated by MAPK in vitro, an event that significantly altered the binding of TRBP to ER and suggests a potential mechanism for regulatory interaction. As the differential recruitment of TRBP to ERalpha and ERbeta may rely on S884, our finding provides insight into estrogen signaling and may lead to the development of therapeutic receptor-selective peptide antagonists.

GT198 Splice Variants Display Dominant-Negative Activities and Are Induced by Inactivating Mutations.

Alternative pre-mRNA splicing yields functionally distinct splice variants in regulating normal cell differentiation as well as cancer development. The putative tumor suppressor gene GT198 (PSMC3IP), encoding a protein also known as TBPIP and Hop2, has been shown to regulate steroid hormone receptor-mediated transcription and to stimulate homologous recombination in DNA repair. Here, we have identified 6 distinct GT198 splice variant transcripts generated by alternative promoter usage or alternative splicing. Various splice variant transcripts preserve a common open reading frame, which encodes the DNA binding domain of GT198. The splice variants act as dominant negatives to counteract wild-type GT198 activity in transcription and to abolish Rad51 foci formation during radiation-induced DNA damage. In fallopian tube cancer, we have identified 44 point mutations in GT198 clustered in 2 mutation hotspot sequences. The mutation hotspots coincide with the regulatory sequences responsible for alternative splicing, strongly supporting that imbalanced alternative splicing is a selected consequence in cancer. In addition, splice variant-associated cytoplasmic expression is found in tumors carrying germline or somatic GT198 mutations. An altered alternative splicing pattern with increased variants is also present in lymphoblastoid cells derived from familial breast cancer patients carrying GT198 germline mutations. Furthermore, GT198 and its variant are reciprocally expressed during mouse stem cell differentiation. The constitutive expression of the GT198 variant but not the wild type induces tumor growth in nude mice. Our results collectively suggest that mutations in the GT198 gene deregulate alternative splicing. Defective alternative splicing promotes antagonizing variants and in turn induces a loss of the wild type in tumorigenesis. The study highlights the role of alternative splicing in tumor suppressor gene inactivation.

Inactivating Mutations in GT198 in Familial and Early-Onset Breast and Ovarian Cancers.

The human GT198 gene (gene symbol PSMC3IP) is located at chromosome 17q21, 470 kb proximal to BRCA1, a locus previously linked to breast and ovarian cancer predisposition. Its protein product (also known as TBPIP and Hop2) has been shown to regulate steroid hormone receptor-mediated gene activation and to stimulate homologous recombination in DNA repair. Here, we screened germline mutations in GT198 in familial and early-onset breast and ovarian cancer patients. We have identified 8 germline variants in a total of 212 index patients including reoccurring nonsense mutation c.310C>T (p.Q104X) and 5' UTR mutation c.-37A>T, each found in 2 unrelated families. Most identified index patients from cancer families had early onsets with a median age of 35 years. c.310C>T was absent in a total of 564 control individuals analyzed. GT198 gene amplification with an imbalanced mutant copy gain was identified in the blood DNA of one of the patients carrying c.310C>T. When tested, this truncating mutation abolished DNA damage-induced Rad51 foci formation. In addition, we have identified 15 somatic mutations in 2 tumors from 1 patient carrying germline mutation c.-37A>T. The presence of a somatic mutation on the wild-type allele showed that GT198 was biallelically mutated in the tumor. The somatic mutations identified near a splicing junction site caused defective alternative splicing and truncated the open reading frame. Therefore, distinct mutations may cause a similar consequence by truncating the full-length protein and inducing a loss of the wild type. Our study provides the first evidence of the presence of inactivating mutations in GT198 in familial and early-onset breast and ovarian cancer patients. Mutations in GT198, a gene regulating DNA repair, potentially contribute to an increased risk in familial breast and ovarian cancers.

Dual roles for coactivator activator and its counterbalancing isoform coactivator modulator in human kidney cell tumorigenesis.

Coactivator activator (CoAA) has been reported to be a coactivator that regulates steroid receptor-mediated transcription and alternative RNA splicing. Herein, we show that CoAA is a dual-function coregulator that inhibits G(1)-S transition in human kidney cells and suppresses anchorage-independent growth and xenograft tumor formation. Suppression occurs in part by down-regulating c-myc and its downstream effectors ccnd1 and skp2 and causing accumulation of p27/Kip1 protein. In this cellular setting, CoAA directly represses the proto-oncogene c-myc by recruiting HDAC3 protein and decreasing both the acetylation of histone H3 and the presence of RNA polymerase II on the c-myc promoter. Interestingly, a splicing isoform of CoAA, coactivator modulator (CoAM), antagonizes CoAA-induced G(1)-S transition and growth inhibition by negatively regulating the mRNA levels of the endogenous CoAA isoform. In addition, we found that expression of CoAA protein is significantly decreased in human renal cell carcinoma compared with normal kidney. Our study presents evidence that CoAA is a potential tumor suppressor in renal carcinoma and that CoAM is a counterbalancing splice isoform. This is, thus far, the only example of a nuclear receptor coregulator involved in suppression of kidney cancer and suggests potentially significant new roles for coregulators in renal cancer biology.

The long terminal repeat (LTR) of ERV-9 human endogenous retrovirus binds to NF-Y in the assembly of an active LTR enhancer complex NF-Y/MZF1/GATA-2.

The solitary ERV-9 long terminal repeat (LTR) located upstream of the HS5 site in the human beta-globin locus control region exhibits prominent enhancer activity in embryonic and erythroid cells. The LTR enhancer contains 14 tandemly repeated subunits with recurrent CCAAT, GTGGGGA, and GATA motifs. Here we showed that in erythroid K562 cells these DNA motifs bound the following three transcription factors: ubiquitous NF-Y and hematopoietic MZF1 and GATA-2. These factors and their target DNA motifs exhibited a hierarchy of DNA/protein and protein/protein binding affinities: NF-Y/CCAAT > NF-Y/GATA-2 > NF-Y/MZF1 > MZF1/GTGGGGA; GATA-2/GATA. Through protein/protein interactions, NF-Y bound at the CCAAT motif recruited MZF1 and GATA-2, but not Sp1 and GATA-1, and stabilized their binding to the neighboring GTGGGGA and GATA sites to assemble a novel LTR enhancer complex, NF-Y/MZF1/GATA-2. In the LTR-HS5-epsilonp-GFP plasmid integrated into K562 cells, mutation of the CCAAT motif in the LTR enhancer to abolish NF-Y binding inactivated the enhancer, closed down the chromatin structure of the epsilon-globin promoter, and silenced transcription of the green fluorescent protein gene. The results indicated that NF-Y bound at the CCAAT motifs assembled a robust LTR enhancer complex, which could act over the intervening DNA to remodel the chromatin structure and to stimulate the transcription of the downstream gene locus.

Nuclear receptor coactivator thyroid hormone receptor-binding protein (TRBP) interacts with and stimulates its associated DNA-dependent protein kinase.

Nuclear receptors mediate gene activation through ligand-dependent interaction with coactivators. We previously cloned and characterized thyroid hormone receptor-binding protein, TRBP (NcoA6: AIB3/ASC-2/RAP250/PRIP/TRBP/NRC), as an LXXLL-containing coactivator that associates with coactivator complexes through its C terminus. To search for protein factors involved in TRBP action, we identified a distinct set of proteins from HeLa nuclear extract that interacts with the C terminus of TRBP. Analysis by mass spectrometric protein sequencing revealed a DNA-dependent protein kinase (DNA-PK) complex including its catalytic subunit and regulatory subunits, Ku70 and Ku86. DNA-PK is a heterotrimeric nuclear phosphatidylinositol 3-kinase that functions in DNA repair, recombination, and transcriptional regulation. DNA-PK phosphorylates TRBP at its C-terminal region, which directly interacts with Ku70 but not Ku86 in vitro. In addition, in the absence of DNA, TRBP itself activates DNA-PK, and the TRBP-stimulated DNA-PK activity has an altered phosphorylation pattern from DNA-stimulated activity. An anti-TRBP antibody inhibits TRBP-induced kinase activity, suggesting that protein content of TRBP is responsible for the stimulation of DNA-independent kinase activity. Furthermore, in DNA-PK-deficient scid cells, TRBP-mediated transactivation is significantly impaired, and nuclear localization of TRBP is altered. The activation of DNA-PK in the absence of DNA ends by the coactivator TRBP suggests a novel mechanism of coactivator-stimulated DNA-PK phosphorylation in transcriptional regulation.

Deletion of the cancer-amplified coactivator AIB3 results in defective placentation and embryonic lethality.

The amplified in breast cancer-3 (AIB3, ASC-2, RAP250, PRIP, TRBP, NRC, or NcoA6) gene is characterized as a cancer-amplified transcriptional coactivator for nuclear receptors, which include the peroxisome proliferator-activated receptor gamma (PPARgamma). To assess its biological function, we deleted the AIB3 gene in mice by homologous recombination. AIB3(+/-) mice are developmentally normal and fertile. AIB3(-/-) embryos exhibit growth restriction and lethality during 9.75-11.5 days postconception. The embryonic lethality is probably attributed to defects in the development of the placental vascular network and cardiac hypoplasia. These defects include the failure of labyrinthine development, the dilation of maternal blood sinuses, the massive erythrophagocytosis by trophoblasts, the alteration of trophoblast populations, and the lower proliferation of myocardium, which are similar to those encountered in mice lacking PPARgamma or the PPARgamma-binding protein (PBP, TRAP220, or DRIP205). In addition, the transcriptional activities of PPARgamma are significantly affected in mouse embryonic fibroblasts lacking AIB3. These results suggest that AIB3 is required for PPARgamma function in placental development and for normal heart development. These results also indicate that the biological function of AIB3 is not redundant with other classes of nuclear receptor coactivators such as PBP and members of the steroid receptor coactivator family.