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The desmoplastic stroma of pancreatic cancers forms a physical barrier that impedes intratumoral drug delivery. Attempts to modulate the desmoplastic stroma to increase delivery of administered chemotherapy have not shown positive clinical results thus far, and preclinical reports in which chemotherapeutic drugs were coadministered with antistromal therapies did not universally demonstrate increased genotoxicity despite increased intratumoral drug levels. In this study, we tested whether TGFβ antagonism can break the stromal barrier, enhance perfusion and tumoral drug delivery, and interrogated cellular and molecular mechanisms by which the tumor prevents synergism with coadministered gemcitabine. TGFβ inhibition in genetically engineered murine models (GEMM) of pancreas cancer enhanced tumoral perfusion and increased intratumoral gemcitabine levels. However, tumors rapidly adapted to TGFβ-dependent stromal modulation, and intratumoral perfusion returned to pre-treatment levels upon extended TGFβ inhibition. Perfusion was governed by the phenotypic identity and distribution of cancer-associated fibroblasts (CAF) with the myelofibroblastic phenotype (myCAFs), and myCAFs which harbored unique genomic signatures rapidly escaped the restricting effects of TGFβ inhibition. Despite the reformation of the stromal barrier and reversal of initially increased intratumoral exposure levels, TGFβ inhibition in cooperation with gemcitabine effectively suppressed tumor growth via cooperative reprogramming of T regulatory cells and stimulation of CD8 T cell-mediated antitumor activity. The antitumor activity was further improved by the addition of anti-PD-L1 immune checkpoint blockade to offset adaptive PD-L1 upregulation induced by TGFβ inhibition. These findings support the development of combined antistroma anticancer therapies capable of impacting the tumor beyond the disruption of the desmoplastic stroma as a physical barrier to improve drug delivery., (©2021 The Authors; Published by the American Association for Cancer Research.)

Beta-carotene 15,15'-monooxygenase (BCM) catalyzes the first step of vitamin A biosynthesis from provitamin A carotenoids. We wished to determine the factors underlying the transcriptional regulation of this gene. After cloning of the 40 kilobase pair (kbp) mouse Bcm gene and determination of its genomic organization, analysis of the 2 kb 5'-flanking region showed several putative transcription factor binding sites including TATA box, a peroxisome proliferator response element (PPRE), AP2, and bHLH. The 2 kb fragment drove specific luciferase gene expression in vitro only in cell lines that express BCM (TC7, PF11, and monkey retinal pigment epithelium). Nucleotides -41 to +163, and -60 to +163 drove basal and specific Bcm transcriptional activity, respectively. Site-directed mutagenesis and gel shift experiments demonstrate that PPRE was essential for Bcm promoter specificity and that the peroxisome proliferator activated receptor (PPAR) gamma (PPARgamma) specifically binds to this element. Furthermore, cotransfection experiments and pharmacological treatments in vitro, using the specific PPARgamma agonists LY17883 and ciglitazone, demonstrate that the PPRE element confers peroxisome proliferator responsiveness via the PPARgamma and retinoid X receptor-alpha heterodimer. Treatment of mice with the PPARalpha/gamma agonist WY14643 increases BCM protein expression in liver. Thus PPAR is a key transcription factor for the transcriptional regulation of the Bcm gene, suggesting a broader function for PPARs in the regulation of carotenoid metabolism metabolism that is consistent with their established role in neutral lipid metabolism and transport.

Annexin A13 (ANXA13) is believed to be the original founder gene of the 12-member vertebrate annexin A family, and it has acquired an intestine-specific expression associated with a highly differentiated intracellular transport function. Molecular characterization of this subfamily in a range of vertebrate species was undertaken to assess coding region conservation, gene organization, chromosomal linkage, and phylogenetic relationships relevant to its progenitor role in the structure-function evolution of the annexin gene superfamily. Protein diagnostic features peculiar to this subfamily include an alternate isoform containing a KGD motif, an elevated basic amino acid content with polyhistidine expansion in the 5'-translated region, and the conservation of 15% core tetrad residues specific to annexin A13 members. The 12 coding exons comprising the 58-kb human ANXA13 gene were deduced from BAC clone sequencing, whereas internal repetitive elements and neighboring genes in chromosome 8q24.12 were identified by contig analysis of the draft sequence from the human genome project. A unique exon splicing pattern in the annexin A13 gene was corroborated by coanalysis of mouse, rat, zebrafish, and pufferfish genomic DNA and determined to be the most distinct of all vertebrate annexins. The putative promoter region was identified by phylogenetic footprinting of potential binding sites for intestine-specific transcription factors. Mouse annexin A13 cDNA was used to map the gene to an orthologous linkage group in mouse chromosome 15 (between Sdc2 and Myc by backcross analysis), and the zebrafish cDNA permitted its localization to linkage group 24. Comparative analysis of annexin A13 from nine species traced this gene's speciation history and assessed coding region variation, whereas phylogenetic analysis showed it to be the deepest-branching vertebrate annexin, and computational analysis estimated the gene age and divergence rate. The unique, conserved aspects of annexin A13 primary structure, gene organization, and genetic maps identify it as the probable common ancestor of all vertebrate annexins, beginning with the sequential duplication to annexins A7 and A11 approximately 700 MYA, before the emergence of chordates.

The regulators of G-protein signaling (RGS) proteins are important regulatory and structural components of G-protein coupled receptor complexes. RGS proteins are GTPase activating proteins (GAPs) of Gi-and Gq-class Galpha proteins, and thereby accelerate signaling kinetics and termination. Here, we mapped the chromosomal positions of all 21 Rgs genes in mouse, and determined human RGS gene structures using genomic sequence from partially assembled bacterial artificial chromosomes (BACs) and Celera fragments. In mice and humans, 18 of 21 RGS genes are either tandemly duplicated or tightly linked to genes encoding other components of G-protein signaling pathways, including Galpha, Ggamma, receptors (GPCR), and receptor kinases (GPRK). A phylogenetic tree revealed seven RGS gene subfamilies in the yeast and metazoan genomes that have been sequenced. We propose that similar systematic analyses of all multigene families from human and other mammalian genomes will help complete the assembly and annotation of the human genome sequence.