Stress and Molecular Drivers for Cancer Progression: A Longstanding Hypothesis.

Stress management is becoming very important part of cancer patient care. Chronic stressors lead to boost tumorigenesis and promote cancer development, recurrence, and drug resistant leading to poor health outcomes. The Hypothalamic-Pituitary-Adrenal (HPA) axis, which is activated by stress, also regulates Hypothalamic-Pituitary-Thyroid (HPT) axis. Stress related changes in immune function and inflammatory response also leads to reduced immune surveillance resulting in tumorigenesis. This article explores the hormonal axis impacted by stress and how chronic stress can lead to poor outcome of a cancer patient.

MUC13 interaction with receptor tyrosine kinase HER2 drives pancreatic ductal adenocarcinoma progression.

Although MUC13, a transmembrane mucin, is aberrantly expressed in pancreatic ductal adenocarcinoma (PDAC) and generally correlates with increased expression of HER2, the underlying mechanism remains poorly understood. Herein, we found that MUC13 co-localizes and interacts with HER2 in PDAC cells (reciprocal co-immunoprecipitation, immunofluorescence, proximity ligation, co-capping assays) and tissues (immunohistofluorescence). The results from this study demonstrate that MUC13 functionally interacts and activates HER2 at p1248 in PDAC cells, leading to stimulation of HER2 signaling cascade, including ERK1/2, FAK, AKT and PAK1 as well as regulation of the growth, cytoskeleton remodeling and motility, invasion of PDAC cells-all collectively contributing to PDAC progression. Interestingly, all of these phenotypic effects of MUC13-HER2 co-localization could be effectively compromised by depleting MUC13 and mediated by the first and second EGF-like domains of MUC13. Further, MUC13-HER2 co-localization also holds true in PDAC tissues with a strong functional correlation with events contributing to increased degree of disorder and cancer aggressiveness. In brief, findings presented here provide compelling evidence of a functional ramification of MUC13-HER2: this interaction could be potentially exploited for targeted therapeutics in a subset of patients harboring an aggressive form of PDAC.

Ubiquitin-like (UBX)-domain-containing protein, UBXN2A, promotes cell death by interfering with the p53-Mortalin interactions in colon cancer cells.

Mortalin (mot-2) induces inactivation of the tumor suppressor p53's transcriptional and apoptotic functions by cytoplasmic sequestration of p53 in select cancers. The mot-2-dependent cytoprotective function enables cancer cells to support malignant transformation. Abrogating the p53-mot-2 interaction can control or slow down the growth of cancer cells. In this study, we report the discovery of a ubiquitin-like (UBX)-domain-containing protein, UBXN2A, which binds to mot-2 and consequently inhibits the binding between mot-2 and p53. Genetic analysis showed that UBXN2A binds to mot-2's substrate binding domain, and it partly overlaps p53's binding site indicating UBXN2A and p53 likely bind to mot-2 competitively. By binding to mot-2, UBXN2A releases p53 from cytosolic sequestration, rescuing the tumor suppressor functions of p53. Biochemical analysis and functional assays showed that the overexpression of UBXN2A and the functional consequences of unsequestered p53 trigger p53-dependent apoptosis. Cells expressing shRNA against UBXN2A showed the opposite effect of that seen with UBXN2A overexpression. The expression of UBXN2A and its apoptotic effects were not observed in normal colonic epithelial cells and p53-/- colon cancer cells. Finally, significant reduction in tumor volume in a xenograft mouse model in response to UBXN2A expression was verified in vivo. Our results introduce UBXN2A as a home defense response protein, which can reconstitute inactive p53-dependent apoptotic pathways. Inhibition of mot-2-p53 interaction by UBXN2A is an attractive therapeutic strategy in mot-2-elevated tumors.

Molecular markers of miscellaneous primary and metastatic tumors of the uterine cervix.

Miscellaneous primary tumors of the uterine cervix are rare. Markers which can be utilized to detect these tumors are very few and in most cases, have not been clinically validated. The information provided in this article will help in developing strategies to discover novel markers and initiate translational research in this ignored area. Based on the reported studies, cytokeratin markers are common in many tumors and few of these rare cancers demonstrate human papilloma-virus (HPV) and Epstein Bar virus (EBV) infection. Due to the very low prevalence of these tumors, epidemiological studies have not been conducted and the etiology of these tumors is largely unknown.

MUC4 expression is regulated by cystic fibrosis transmembrane conductance regulator in pancreatic adenocarcinoma cells via transcriptional and post-translational mechanisms.

MUC4 mucin is a high molecular weight transmembrane glycoprotein that plays important roles in tumour biology. It is aberrantly expressed in pancreatic adenocarcinoma, while not being detectable in the normal pancreas. Previous studies have demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that is defective in CF, is implicated in multiple cellular functions, including gene regulation. In the present study, using a CFTR-defective pancreatic cancer cell line and its derived subline expressing functional CFTR, we report that MUC4 expression is negatively regulated by CFTR. Short-interfering RNA (siRNA)-mediated silencing of CFTR also leads to an increased expression of MUC4. Additionally, our results suggest that CFTR-mediated regulation of MUC4 is cell density-dependent and is achieved by transcriptional and posttranslational mechanisms. Moreover, in a panel of pancreatic cancer cell lines and normal pancreas, we observed that CFTR was downregulated in pancreatic cancer cells and negatively correlated with MUC4 in most cases. An aberrant expression of MUC4 was also detected in the CF pancreas. Downregulation of CFTR in pancreatic adenocarcinoma and its inverse association with the tumour-linked mucin, MUC4, indicate novel function(s) of CFTR in pancreatic tumour biology and suggest the implication of new signalling pathway(s) in MUC4 regulation.

The human homologue of the RNA polymerase II-associated factor 1 (hPaf1), localized on the 19q13 amplicon, is associated with tumorigenesis.

The 19q13 amplicon in pancreatic cancer cells contains a novel pancreatic differentiation 2 (PD2) gene (accession number AJ401156), which was identified by differential screening analysis. PD2 is the human homologue of the RNA polymerase II-associated factor 1 (hPaf1). In yeast, Paf1 is part of the transcription machinery, acting as a docking protein in between the complexes Rad6-Bre1, COMPASS-Dot1p, and the phosphorylated carboxyl terminal domain of the RNA polymerase II. As such, Paf1 is directly involved in transcription elongation via histone H2B ubiquitination and histone H3 methylation. The PD2 sequence is highly conserved from Drosophila to humans with up to 98% identity between rodent and human, suggesting the functional importance of PD2/hPaf1 to maintain cellular homeostasis. PD2 is a modular protein composed of RNA recognition motif, DEAD-boxes, an aspartic/serine (DS)-domain, a regulator of the chromosome condensation domain and myc-type helix-loop-helix domains. Our results further showed that PD2 is a nuclear 80 kDa protein, which interacts with RNA polymerase II. In addition, we have demonstrated that the overexpression of PD2 in the NIH 3T3 cells result in enhanced growth rates in vitro and tumor formation in vivo. Altogether, this paper presents strong evidence that the overexpression of PD2/hPaf1 is involved in cancer development.

Monoclonal antibody against human sperm-specific YLP12 peptide sequence involved in oocyte binding.

The monoclonalantibody (mAb) against YLP12 peptide was raised and its immunobiological properties were examined. In the Western blot procedure, the YLP12 mAb recognized a specific protein band of approximately 50 +/- 5 kD in human sperm extract and approximately 72 +/- 5 kD in human testis extract. The myeloma Ig control did not recognize these specific protein bands. In the immunofluorescence studies, the YLP12 mAb, and not the myeloma Ig, predominantly reacted with the acrosome regions of methanol-fixed human sperm. In the acrosome reaction assay, the YLP12 mAb showed a significant (p < .001) and a concentration-dependent inhibition of acrosome reaction. The myeloma Ig did not affect the acrosome reaction. There was no apparent effect of antibodies on sperm motility. Thus, the monoclonal antibody, if humanized by genetic engineering technology, may provide a useful immunocontraceptive agent.

Effect of antibodies to sperm-specific recombinant contraceptive vaccinogen (rCV) on murine fertilization: search for an animal model to examine its contraceptive potential.

Recently, we cloned and sequenced a sperm-specific antigen, designated as Contraceptive Vaccinogen (rCV), from human testis (Naz et al., 2001). The present study was conducted to examine its proteomic homologue and function in murine sperm, in order to find out whether or not the mouse can provide a suitable model for examining its immunocontraceptive effects. This was examined by using purified antibodies (Ab) raised against the recombinant (r) human CV antigen of approximately 44 kD. In the Western blot procedure, rCV antibodies recognized a specific protein band of approximately 64 +/- 5 kD in murine testis and murine sperm extracts, the band similar to that found in human testis and human sperm. In the immunoprecipitation procedure, rCV Ab immunoprecipitated a protein band of similar size from murine sperm and murine testis extracts. The immunocytochemical (ICT), immunoscanning electronmicroscopic (ISEM) and the immunobead binding technique (IBT) revealed the subcellular localization of CV antigen on the surface of acrosome and tail regions of the noncapacitated and capacitated murine sperm cell. In functional bioassays, rCV Ab inhibited the acrosome reaction as well as sperm-egg binding in vitro. These data indicate that the CV antigen is expressed in murine sperm and has a biological role in sperm function and sperm-egg binding. In vitro inhibition of capacitation/acrosome reaction and sperm-zona binding suggest that the mouse can provide a suitable model to examine the immunocontraceptive effects of CV antigen in actively-immunized animals.

Presence of antibodies to sperm YLP(12) synthetic peptide in sera and seminal plasma of immunoinfertile men.

Using an enzyme-linked immunosorbent assay (ELISA), sera (n = 15) and seminal plasma (n = 30) from antisperm antibody-positive immunoinfertile men (n = 45) and from fertile men (n = 45), were tested for the immunoreactivity with the synthetic YLP(12) sperm peptide. Of the 15 immunoinfertile sera tested, 46% were positive for immunoglobulin (Ig)M, 73% for IgG, and 40% for IgA. Of the 30 samples of immunoinfertile seminal plasma tested, 10% were positive for IgM, 20% for IgG, and 43% for IgA. None of the sera or seminal plasma from fertile men showed a positive reaction. There was no significant correlation between the sperm immobilization technique (SIT) or tray agglutination technique (TAT) titres or percentage binding in immunobead binding technique (IBT) and the antibody reactivity for any class in the ELISA. The YLP(12) peptide conjugated to bovine serum albumin-Sepharose 4B beads pulled out IgG antibodies from the serum of the immunoinfertile, but not the fertile, men. The beads pulled out IgA antibodies from the immunoinfertile, but not the fertile, seminal plasma. The immuno-affinity purified antipeptide antibodies reacted with a specific band of 72 +/- 5 kDa in the human testis and with a specific band of approximately 50 +/- 5 kDa in the human sperm extracts. The YLP(12) peptide may have applications in the specific diagnosis and treatment of male infertility and in contraceptive vaccine development.

Expression of alpha and gamma interferon receptors in the sperm cell.

The present study was conducted to examine whether or not the sperm cell has the expression of receptors for interferon (IFN) -alpha and -gamma. This was investigated using specific antibodies. Antibody to IFN-alpha receptor reacted with the acrosomal and tail regions of the murine sperm cell in the indirect immunofluorescence technique (IFT) and immunoscanning electron microscopic procedure (ISEP). In the immunoprecipitation and Western blot procedures, this antibody specifically recognized a protein band of approximately 100 kD, which corresponds to the molecular weight of IFN-alpha receptor present in other cell types. Antibody to IFN-gamma receptor specifically reacted with the posterior head, midpiece, and tail regions of sperm cell in IFT and ISEP, and recognized a band of approximately 85 kD in the immunoprecipitation and Western blot procedures, corresponding to the IFN-alpha receptor. Similar bands of approximately 100 kD and approximately 85 kD molecular identities were also detected in the testes extracts and sperm extracts of other mammalian species namely human, rabbit, and pig, the species tested. These findings indicate that the mammalian sperm cell has expression of IFN-alpha and IFN-gamma receptors, which seem to develop during spermatogenesis in the testes. These findings may have implications in male infertility and antisperm contraceptive vaccine development.

Uterine luminal epithelial alkaline phosphatase activity and pinopod development in relation to endometrial sensitivity in the rat.

The period of maximal endometrial sensitivity in the rat was characterized by high alkaline phosphatase activity in uterine luminal and glandular epithelium and endometrial stroma. The activity in endometrial stroma increased following decidualization. Pinopod development on the endometrial surface was first observed during the presensitivity period. Their number increased, apparently more so on the antimesometrial rather than the mesometrial segment of the uterus, on the day of maximal sensitivity. Inhibition in endometrial sensitivity by single anti-implantation (1.25 mg/kg, po) dose of centchroman on day 1 post-coitum (p.c.), although it did not affect alkaline phosphatase activity on days 2 and 3 p.c., caused complete inhibition in its activity in uterine luminal and glandular epithelium and pinopod development on days 4 and 5-coinciding, respectively, with time of entry of preimplantation embryos into the uterus and period of maximal endometrial sensitivity in this species. Significant decrease in enzyme activity was also evident in the entire endometrial stroma and myometrium, except blood capillaries, on these days. In comparison, prevention of entry of native embryos into the uterus by placing a ligature at the utero-tubal junction had no effect on pinopod development, but caused marked decrease in enzyme activity in luminal and glandular epithelium only during the immediate postimplantation period. The uterine lumen on the day of maximal sensitivity in centchroman-treated rats appeared highly distended and was lined with tall columnar epithelium, in comparison to low cuboidal epithelium in controls. The findings demonstrate: (a) a correlation between uterine luminal epithelial alkaline phosphatase activity and endometrial sensitivity; (b) complete inhibition in enzyme activity in luminal and glandular epithelium following centchroman treatment might be related to altered permeability characteristics of epithelial cells, which together with the absence of pinopods and highly distended uterine lumen on the day of maximal sensitivity, suggest inhibition of endocytosis/pinocytosis of luminal fluid, luminal closure, apposition of blastocyst trophoblast to luminal epithelium, and secretory activity of glandular epithelium; (c) pinopod development on the endometrial surface was independent of presence of viable blastocysts in utero; and (d) complete absence of pinopods suggests lack of endometrial sensitivity, but their presence might not necessarily indicate a sensitized endometrium in the rat.

Inhibition of progesterone-induced development of giant mitochondria in uterine glandular epithelial cells by an antiestrogen in rat.

Effect of antiestrogen ormeloxifene on progesterone-induced development of giant mitochondria in uterine glandular epithelial cells in ovariectomized adult Sprague-Dawley rats was investigated. Antiestrogen tamoxifen and antiprogestin onapristone were used for comparison. Well-formed giant mitochondria were observed in uterine glandular epithelial cells in rats treated with progesterone (20 mg/kg, subcutaneous [SC] per se for three consecutive days. In rats primed with estradiol-17 beta (0.5 micrograms/day, s.c.) on days-2 and -1 before progesterone treatment, there was, in addition, a marked increase and distension of smooth endoplasmic reticulum (SER) and Golgi complex, in comparison to extensive rough endoplasmic reticulum (RER) and polyribosomes in progesterone per se treated rats. Cytological picture in rats receiving single (1.25 mg/kg, day 1, p.o.) or multiple (0.25 mg/kg, days 1-3, p.o.) anti-implantation doses of ormeloxifene showed marked reduction in glandular epithelial cell height, luminal surface microvilli, RER, polyribosomes and cytoplasm:nucleus ratio, straightening of intercellular membranes, and interdigitation of basement membrane. Mitochondria were of normal size and nuclei were heterochromatic. Inhibition in the case of tamoxifen (0.1 mg/kg, days 1-3, p.o.) appeared partial and rare giant mitochondria with degenerating cristae and outer membranes were apparent. Onapristone (10 mg/kg, day 1, s.c.), in addition to inhibiting development of giant mitochondria, caused extensive vacuolization and distension of intercellular membranes in glandular epithelial cells.

Uterine estradiol and progesterone receptor concentration, activities of certain antioxidant enzymes and dehydrogenases and histoarchitecture in relation to time of secretion of nidatory estrogen and high endometrial sensitivity in rat.

Alterations in uterine nuclear and cytosolic estradiol (ER) and progesterone (PR) receptor concentration, activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glucose-6-phosphate dehydrogenase (G-6-PDH) and lactate dehydrogenase (LDH), surface and transmission electron microscopy and histology in relation to the time of secretion of nidatory estrogen and the onset of endometrial sensitivity in the rat were investigated. A significant increase in plasma estradiol (E2) concentration in control rats was observed at 22.00 h on day 4 post-coitum, whereas progesterone (P) concentration increased at 17.00 h on day 4 and was maintained until 17.00 h on day 5. The period of high endometrial sensitivity (10.00 h on day 5) was characterized by elevated uterine cytosolic ER and nuclear and cytosolic PR concentration and POD activity, low columnar luminal epithelium with undulating surface and intercellular membranes, covered with short microvilli and pinopods, and containing numerous electron-transparent apical vesicles, mitochondria, polyribosomes, rough (RER) and smooth (SER) endoplasmic reticulum, well developed Golgi, few lysosomes and lipid droplets and loose edematous antimesometrial stroma. Inhibition in endometrial sensitivity by post-coital centchroman was associated with a marked depletion in uterine cytosolic ER and an increase in nuclear ER concentration, a decrease in POD and G-6-PDH activities, compact fibroblastic stroma, an increase in luminal epithelial cell height with decreased RER, SER, polyribosomes, Golgi, straightening of intercellular membranes, reduced surface undulations and absence of pinopods. Electron-transparent vesicles appeared flattened and clumped in the apical portion of cells, tight junctions were more prominent and lipid droplets were translucent. Nuclear and cytosolic PR and the pattern of secretion or plasma E2 and P remained unaffected. CAT, SOD and LDH activities, although high throughout pre-implantation, did not vary in relation to the secretion of nidatory estrogen, endometrial sensitivity or centchroman treatment.

Correlation of pinopod development on uterine luminal epithelial surface with hormonal events and endometrial sensitivity in rat.

Intrinsic role of preovulatory and nidatory estrogen and progesterone and presence of viable blastocysts in utero in pinopod development on the uterine luminal epithelial surface and correlation between time of their development and onset of endometrial sensitivity were investigated. In adult rats, pinopods were observed on the entire epithelium even before secretion of nidatory estrogen, i.e. at 14.00 h on day 4 post-coitum (p.c.). Apparently, their number increased, more so on the antimesometrial than the mesometrial side, at 10.00 h on day 5, but were fewer and mostly collapsed at 10.00 h on day 6. Pinopods on day 4 were located within epithelial depressions and foldings, but protruded from the surface on days 5 and 6. Normal pinopods were also present on day 8 p.c. in rats under delayed implantation, but an implantation-inducing dose of estradiol-17 beta administered about 18 h earlier caused their collapse like that on day 6 in intact rats. Development and appearance of pinopods in intact or delayed rats was unaffected when native preimplantation embryos were prevented from entering the uterus. Normal pinopods were seen in immature rats receiving progesterone for at least 3 days or cyproterone acetate for 4 days, but not after estradiol alone. In animals receiving progesterone or priming/sensitizing estradiol in addition to progesterone, the decidual response was suboptimal, irrespective of the presence of pinopods on the day of stimulation. In animals in which a condition mimicking preimplantation had been produced by suitable hormone supplementation, optimal endometrial sensitivity and decidual response were elicited, even though most pinopods appeared collapsed, resembling those on day 6 in intact rats and about 18 h after estradiol in implantation-delayed rats. Findings confirm that pinopod development on uterine luminal epithelium was dependent on progesterone alone and demonstrate that: (i) preovulatory (priming) or nidatory (endometrial sensitizing) estrogen or viable blastocysts in utero have no role in their development. Nidatory estrogen, instead, appears to limit pinopod development by causing their collapse; (ii) pinopod development/presence on the endometrial surface might indicate the uterus coming into a period of sensitivity rather than actually being in it and might thus serve as a useful marker of "transfer window" rather than "implantation window"; (iii) in the rat, pinopod development might serve as an alternate assay for evaluation of progestational activity of newer test agents.

Time-related effects of a triphenylethylene antiestrogen on estrogen-induced changes in uterine weight, estrogen receptors, and endometrial sensitivity in rats.

Time-related estrogen antagonistic action of a single oral contraceptive (1.25 mg/kg) dose of the triphenylethylene antiestrogen centchroman was determined in ovariectomized immature rats. Tamoxifen and nafoxidine were used for comparison. A single oral administration of centchroman followed by three doses of estradiol-17 beta (1 microgram/d, s.c.) caused significant dose-dependent inhibition in estradiol-17 beta-induced increase in uterine weight and nuclear and cytosolic estrogen receptors. But the inhibition at antiimplantation dose was evident only if estradiol-17 beta treatment was initiated not later than 48 h post-antiestrogen. Alternatively, when antiestrogen treatment was followed by a single dose of estradiol-17 beta between days 2-7, a synergistic action, typical of antiestrogens possessing weak estrogen agonistic activity, was observed. In immature rats in which a condition mimicking preimplantation was produced by estradiol-17 beta (0.5 microgram/d, s.c.) priming on days -2 and -1, followed by progesterone (1 mg/d, s.c.) and an endometrial sensitizing dose (0.5 microgram/d, s.c.) of estradiol-17 beta at 1600 h on day 4, anti-implantation dose of centchroman administered on day 1, too, failed to inhibit uterine weight gain induced by sensitizing dose of estradiol-17 beta, but caused marked inhibition in endometrial sensitivity to a deciduogenic stimulus and decidualization and weight gain of traumatized uterine horn 96 h post-traumatization over non-traumatized horn was only about 150% (725% in controls). Inhibition in endometrial sensitivity and decidualization was evident when the interval between antiestrogen treatment and sensitizing estradiol was < 126 h. Pinopods were present on endometrial surface on day 5 whether or not priming and/or sensitizing doses of estradiol were administered, but decidual response was mild if either of these doses of estradiol-17 beta was deferred. Findings suggest that: (a) duration of antiestrogenic action of single anti-implantation dose of centchroman in rat was about 126 h, which in ovariectomized immature rats was evident only when a condition mimicking preimplantation was produced and the antiestrogenic response was based on inhibition in estradiol-induced endometrial sensitivity and not uterine weight gain; (b) priming as well as sensitizing estrogen were essential to get optimal decidual responses; (c) appearance of pinopods on endometrial surface may not be related to endometrial sensitivity; and (d) tamoxifen and nafoxidine appear slightly longer acting with duration of antiestrogenic action of approximately 150 h.