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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2011)

Tumor formation/progression is determined by a wide array of factors including downregulation of tumor suppressors and upregulation of oncogenes. In breast cancers, mutation of the tumor suppressor, breast cancer 1 (BRCA1), accounts for the majority of inherited cases, which themselves account for about 10% of the total. Given the importance of BRCA1 in the development of breast cancer and the fact that the majority of breast cancers have wild type BRCA1, the question posed in the current study was what happens to BRCA1 tumor suppressor activity during the development of most breast cancers. To examine this issue, we employed two forms of prolactin that work through the same receptors, differ from one another only by a single amino acid, and have antagonistic activity in terms of cell proliferation and survival. Also, since BRCA1 mutations have recently been shown to be important in ovarian and prostate cancer development, the study additionally included cell lines representative of these cancers. Results show that wild type BRCA1 could be functionally silenced by prolactin in terms of its ability to induce transcription of the cell cycle inhibitor, p21, whereas the prolactin antagonist increased expression of p21 through BRCA1. The functional silencing in response to prolactin occurred as a result of an interaction with the signaling molecule, Stat5 and could be blocked by a dominant negative Stat5. In addition, we showed that prolactin also contributes to tumor progression by regulating p21 at post-transcriptional and post-translational stages. The post-transcriptional regulation was mediated by miRNA. One candidate for this activity is miR-106, a miRNA induced by PRL, likely through an estrogen receptor α (ERα) and c-myc pathway. Prolactin also induced phosphorylation of p21 on threonine 145, causing it to be retained in the cytosol under which circumstances it inhibits apoptosis. At present, the kinase responsible for prolactin-stimulated phosphorylation of p21 is unclear, but preliminary data suggest a possible role for pim-1.

The prolactin antagonist employed in these studies is a molecular mimic of naturally phosphorylated prolactin. Given its ability to increase expression of p21 and antagonize the effects of unmodified prolactin, one would predict that the ratio of unmodified prolactin to phosphorylated prolactin may be important to the development/progression of cancer. To test this in a clinical setting, a serum-based assay capable of quantifying phosphorylated prolactin as a separate entity from unmodified prolactin is required. To develop such an assay, regions of the p21 promoter were deleted to determine a minimal promoter responsive to phosphorylated prolactin. A 143bp region containing only the BRCA1 response element and TATA box was determined to be sufficient and specific to the phosphorylated form of the hormone. Establishment of stable cell lines expressing this minimal promoter linked to luciferase has shown the resultant bioassay to be sensitive and consistent.

    Cover page: Regulation of BRCA1/p21 Axis by Prolactin
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2019)
    Small oligonucleotides (oligos) are increasingly being utilized as diagnostics or treatments for disease. An impediment to broader use is the ability to readily measure oligos in biological fluids. Here, we describe a very straightforward assay with detection in the sub-picomole range that does not require extraction from serum/plasma or polymerization chain reaction amplification. As a result, there are no losses or errors due to sample handling, and the assay can be used to measure oligos modified in a variety of ways that increase therapeutic efficacy. The enzyme-linked oligonucleotide hybridization assay (ELOHA) is based on competition with a detection oligo for hybridization to a capture oligo covalently linked to a solid substrate. The versatility of ELOHAs is demonstrated by application to the measurement of three oligos, including two morpholino-oligos with 3'-octaguanidine derivatization for efficient cell uptake. The third oligo is unmodified and has a DNA sequence equivalent to miR93. The assays have sensitivity as low as 0.28 pmol/sample reaction at 50% hybridization. Adding to clinical utility is the need for only a simple 96-well absorbance plate reader and the finding that neither EDTA nor heparin interferes with detection.
      Cover page: Enzyme-linked oligonucleotide hybridization assay for direct oligo measurement in blood
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2021)
      The mechanisms whereby obesity differentially affects males and females are unclear. Because macrophages are functionally the most important cells in obesity-induced inflammation, we sought to determine reasons for male-specific propensity in macrophage migration. We previously determined that male mice fed a high-fat diet exhibit macrophage infiltration into the hypothalamus, whereas females were protected irrespective of ovarian estrogen, in this study, we show that males accumulate more macrophages in adipose tissues that are also more inflammatory. Using bone marrow cells or macrophages differentiated in vitro from male and female mice fed control or high-fat diet, we demonstrated that macrophages derived from male mice are intrinsically more migratory. We determined that males have higher levels of leptin in serum and adipose tissue. Serum CCL2 levels, however, are the same in males and females, although they are increased in obese mice compared with lean mice of both sexes. Leptin receptor and free fatty acid (FFA) receptor, GPR120, are upregulated only in macrophages derived from male mice when cultured in the presence of FFA to mimic hyperlipidemia of obesity. Unless previously stimulated with LPS, CCL2 did not cause migration of macrophages. Leptin, however, elicited migration of macrophages from both sexes. Macrophages from male mice maintained migratory capacity when cultured with FFA, whereas female macrophages failed to migrate. Therefore, both hyperlipidemia and hyperleptinemia contribute to male macrophage-specific migration because increased FFA induce leptin receptors, whereas higher leptin causes migration. Our results may explain sex differences in obesity-mediated disorders caused by macrophage infiltration.
        Cover page: Sex Differences in Macrophage Responses to Obesity-Mediated Changes Determine Migratory and Inflammatory Traits
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2021)

        Background

        Obesity is characterized by a systemic inflammation and hypothalamic neuroinflammation. Systemic inflammation is caused by macrophages that infiltrate obese adipose tissues. We previously demonstrated that high-fat diet (HFD)-fed male mice exhibited peripheral macrophage infiltration into the hypothalamus, in addition to activation of resident microglia. Since this infiltration contributes to neuroinflammation and neuronal impairment, herein we characterize the phenotype and origin of these hypothalamic macrophages in HFD mice.

        Methods

        C57BL/6J mice were fed HFD (60% kcal from fat) or control diet with matching sucrose levels, for 12-16 weeks. Males and females were analyzed separately to determine sex-specific responses to HFD. Differences in hypothalamic gene expression in HFD-fed male and female mice, compared to their lean controls, in two different areas of the hypothalamus, were determined using the NanoString neuroinflammation panel. Phenotypic changes in macrophages that infiltrated the hypothalamus in HFD-fed mice were determined by analyzing cell surface markers using flow cytometry and compared to changes in macrophages from the adipose tissue and peritoneal cavity. Adipose tissue transplantation was performed to determine the source of hypothalamic macrophages.

        Results

        We determined that hypothalamic gene expression profiles demonstrate sex-specific and region-specific diet-induced changes. Sex-specific changes included larger changes in males, while region-specific changes included larger changes in the area surrounding the median eminence. Several genes were identified that may provide partial protection to female mice. We also identified diet-induced changes in macrophage migration into the hypothalamus, adipose tissue, and peritoneal cavity, specifically in males. Further, we determined that hypothalamus-infiltrating macrophages express pro-inflammatory markers and markers of metabolically activated macrophages that were identical to markers of adipose tissue macrophages in HFD-fed mice. Employing adipose tissue transplant, we demonstrate that hypothalamic macrophages can originate from the visceral adipose tissue.

        Conclusion

        HFD-fed males experience higher neuroinflammation than females, likely because they accumulate more visceral fat, which provides a source of pro-inflammatory macrophages that migrate to other tissues, including the hypothalamus. Our findings may explain the male bias for neuroinflammation and the metabolic syndrome. Together, our results demonstrate a new connection between the adipose tissue and the hypothalamus in obesity that contributes to neuroinflammation and hypothalamic pathologies.
          Cover page: Visceral adipose tissue imparts peripheral macrophage influx into the hypothalamus.
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2017)
          Prolactin promotes a variety of cancers by an array of different mechanisms. Here, we have investigated prolactin's inhibitory effect on expression of the cell cycle-regulating protein, p21. Using a miRNA array, we identified a number of miRNAs upregulated by prolactin treatment, but one in particular that was strongly induced by prolactin and predicted to bind to the 3'UTR of p21 mRNA, miR-106b. By creating a p21 mRNA 3'UTR-luciferase mRNA construct, we demonstrated degradation of the construct in response to prolactin in human breast, prostate and ovarian cancer cell lines. Increased expression of miR-106b replicated, and anti-miR-106b counteracted, the effects of prolactin on degradation of the 3'UTR construct, p21 mRNA levels, and cell proliferation in breast (T47D) and prostate (PC3) cancer cells. Increased expression of miR-106b also stimulated migration of the very epithelioid T47D cell line. By contrast, anti-miR-106b dramatically decreased expression of the mesenchymal markers, SNAIL-2, TWIST-2, VIMENTIN, and FIBRONECTIN. Using signaling pathway inhibitors and the 3'UTR construct, induction of miR-106b by prolactin was determined to be mediated through the MAPK/ERK and PI3K/Akt pathways and not through Jak2/Stat5 in both T47D and PC3 cells. Prolactin activation of MAPK/ERK and PI3K/Akt also activates ERα in the absence of an ERα ligand. 17β-estradiol promoted degradation of the construct in both cell lines and pre-incubation in the estrogen antagonist, Fulvestrant, blocked the ability of both prolactin and 17β-estradiol to induce the construct-degrading activity. Together, these data support a convergence of the prolactin and 17β-estradiol miR-106b-elevating signaling pathways at ERα.
            Cover page: Involvement of miR-106b in tumorigenic actions of both prolactin and estradiol
            • Article
            • Peer Reviewed
            UC Riverside Previously Published Works (2018)
            Matrix metalloproteinases (MMPs) are considered excellent targets for cancer therapy because of their important roles in multiple aspects of tumor growth and metastatic spread. However, not all MMPs, or even all activities of specific MMPs, promote cancer. Therefore, there is a need for highly specific inhibitors. Monoclonal antibodies provide the potential for the degree of specificity required, but the isolation of antibodies able to inhibit a specific protease with high selectivity is challenging. Proteolysis specificity lies in recognition of the substrate in or around the active site, which generally forms a concave cleft inaccessible by human IgGs. Inspired by camelid antibodies, which have convex paratopes, we have produced a recombinant human IgG, designated 3A2, which binds in the substrate cleft of MMP-14, inhibiting its activity, but not the activity of highly homologous MMPs. In the 4T1 highly metastatic, syngeneic, orthotopic model of breast cancer, IgG 3A2 markedly inhibited growth of the primary tumor, but more importantly reduced metastatic spread to the lungs and liver by 94%. Stem cells in the tumor population expressed twice as much MMP-14 mRNA as bulk tumor cells. In addition to reducing dissemination of tumor stem cells, as would be expected from inhibition of MMP-14's ability to degrade components of the extracellular matrix, IgG 3A2 also inhibited the ability of individual stem cells to proliferate and produce colonies. We conclude that it is possible to produce antibodies with sufficient specificity for development as therapeutics and that IgG 3A2 has therapeutic potential.
              Cover page: Use of a novel camelid-inspired human antibody demonstrates the importance of MMP-14 to cancer stem cell function in the metastatic processCreative Commons 'BY-NC' version 4.0 license
              • Article
              • Peer Reviewed
              UC Riverside Previously Published Works (2018)
              Targeting effectual epitopes is essential for therapeutic antibodies to accomplish their desired biological functions. This study developed a competitive dual color fluorescence-activated cell sorting (FACS) to maturate a matrix metalloprotease 14 (MMP-14) inhibitory antibody. Epitope-specific screening was achieved by selection on MMP-14 during competition with N-terminal domain of tissue inhibitor of metalloproteinase-2 (TIMP-2) (nTIMP-2), a native inhibitor of MMP-14 binding strongly to its catalytic cleft. 3A2 variants with high potency, selectivity, and improved affinity and proteolytic stability were isolated from a random mutagenesis library. Binding kinetics indicated that the affinity improvements were mainly from slower dissociation rates. In vitro degradation tests suggested the isolated variants had half lives 6-11-fold longer than the wt. Inhibition kinetics suggested they were competitive inhibitors which showed excellent selectivity toward MMP-14 over highly homologous MMP-9. Alanine scanning revealed that they bound to the vicinity of MMP-14 catalytic cleft especially residues F204 and F260, suggesting that the desired epitope was maintained during maturation. When converted to immunoglobulin G, B3 showed 5.0 nM binding affinity and 6.5 nM inhibition potency with in vivo half-life of 4.6 days in mice. In addition to protease inhibitory antibodies, the competitive FACS described here can be applied for discovery and engineering biosimilars, and in general for other circumstances where epitope-specific modulation is needed.
                Cover page: Epitope‐specific affinity maturation improved stability of potent protease inhibitory antibodies
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