Morphology of Tissue Disruption at Sites of High-Grade Tumors.

BACKGROUND: Invasive solid cancers originating from diverse organs like breast, ovary and lung metastasize to distant sites. The structural changes at the primary sites of these high-grade tumors have not been well characterized. The main aim of the current study was to examine if there is any morphological overlap of metastasizing tissues of different invasive tumors. METHODS: Whole slide hematoxylin and eosin (H&E) stained images from web repository of multiple tumor specimens were used for this study. ImageJ was used for image processing and analysis. RESULTS: The metastatic tissue(s) at the primary sites of different examined high-grade tumors appeared similar, irrespective of the organ of origin of the primary tumor. Numerous cellular excrescences with the repetitive appearance of a bulb-like projection with a narrowed-off trailing end were seen emanating from the tumor cell membrane. Many of them contained nuclei, while others were empty vesicles. Although these structures were not exactly equal in their dimensions, the rubrics of architectural distortion in different high-grade tumors were conserved. CONCLUSIONS: The preliminary observations suggest for the first time that there is structural similarity of the epithelial dysmorphia in many high-grade invasive tumors irrespective of their parental tissue of origin. This commonality of morphological prints of metastases suggests that common pathways of cytosolic force generation are activated during temporal progression of cancer, resulting in the conserved mushroom-shaped appearance of the dismantling individual cell or cell clusters from the parental epithelium. The conserved genomic mechanisms underlying these fascinating observations merit testing and validation in future studies.

MicroRNA profiles were altered in neonatal piglet mammary glands following postnatal infant formula feeding.

Postnatal dietary modulation of microRNAs (miRNAs) and effects on miRNA-mRNA interactions in tissues remain unknown. This study aimed to investigate whether dietary factors (formula vs. breastfeeding) affect mammary miRNA expression and to determine if these changes are concurrent with developmental alterations of the mammary gland in neonatal piglets. Female Yorkshire/Duroc piglets were fed sow's milk or cow's milk- or soy-based infant formula (from postnatal day 2 to day 21; n=6/group). Differentially expressed miRNAs were determined using mammary miRNA profiling, followed by miRNA and mRNA expressions characterized by quantitative reverse-transcription polymerase chain reaction. Milk and soy formulas reduced expressions of miR-1, -128, -133a, -193b, -206 and -27a; miRNA down-regulation altered mRNA expressions of genes (e.g., Ccnd1, Tgfb3, Igf1r and Tbx3) that were consistent with enhanced cell proliferation and suppressed apoptotic processes in the developing mammary gland. Interestingly, down-regulation of miR-1, -128 and -27a also correlated with increased mRNA genes such as Hmgcs and Hmgcr encoding cholesterol synthesis in the mammary glands in response to lower circulating cholesterol levels. Infant formula feeding affected mammary miRNA profiles in neonatal piglets, concurrent with increased expression of cell proliferation and cholesterol synthesis genes, suggesting early nutritional modulation of miRNAs may contribute to regulation of proliferative status and cholesterol homeostasis of developing mammary glands during infancy.

Infant Formula Feeding Changes the Proliferative Status in Piglet Neonatal Mammary Glands Independently of Estrogen Signaling.

BACKGROUND: Soy infant formula contains isoflavones, which are able to bind to and activate estrogen receptor (ER) pathways. The mammary gland is sensitive to estrogens, raising concern that the use of soy formulas may promote premature development. OBJECTIVE: We aimed to determine if soy formula feeding increases mammary gland proliferation and differentiation in comparison to other infant postnatal diets. METHODS: White-Dutch Landrace piglets aged 2 d received either sow milk (Sow), or were provided milk formula (Milk), soy formula (Soy), milk formula supplemented with 17-beta-estradiol (2 mg/(kg·d); M + E2), or milk formula supplemented with genistein (84 mg/L of diet; M + G) until day 21. Mammary gland proliferation and differentiation was assessed by histology, and real-time RT-PCR confirmation of differentially expressed genes identified by microarray analysis. RESULTS: Mammary terminal end bud numbers were 19-31% greater in the Milk, Soy, and M + G groups relative to the Sow and M + E2, P <0.05. Microarray analysis identified differentially expressed genes between each formula-fed group relative to the Sow (±1.7-fold, P <0.05). Real-time RT-PCR confirmed 2- to 4-fold increases in mRNA transcripts of genes involved in cell proliferation, insulin-like growth factor 1 (IGF1), fibroblast growth factor 10 (FGF10), and fibroblast growth factor 18 (FGF18), in all groups relative to the Sow, P <0.05. In contrast, genes involved in cell differentiation and ductal morphogenesis, angiotensin II receptor type 2 (AGTR2), microtubule associated protein 1b (MAP1B), and kinesin family member 26b (KIF26B), were significantly upregulated by 2-, 4-, and 13-fold, respectively, in the M + E2 group. Additionally, mRNA expression of ER-specific gene targets, progesterone receptor (PGR), was increased by 12-fold, and amphiregulin (AREG) and Ras-like estrogen regulated growth inhibitor (RERG) expression by 1.5-fold in the M + E2 group, P <0.05. In the soy and M + G groups, mRNA expressions of fatty acid synthesis genes were increased 2- to 4-fold. CONCLUSIONS: Our data indicate soy formula feeding does not promote ER-signaling in the piglet mammary gland. Infant formula feeding (milk- or soy-based) may initiate proliferative pathways independently of estrogenic signaling.

HSV1/2 Genital Infection in Mice Cause Reversible Delayed Gastrointestinal Transit: A Model for Enteric Myopathy.

In an interesting investigation by Khoury-Hanold et al. (1), genital infection of mice with herpes simplex virus 1 (HSV1) were reported to cause multiple pelvic organ involvement and obstruction. A small subset of mice succumbed after the first week of HSV1 infection. The authors inferred that the mice died due to toxic megacolon. In a severe form of mechanical and/or functional obstruction involving gross dilation of the colon and profound toxemia, the presentation is called "toxic megacolon." The representative observations by Khoury-Hanold likely do not resemble toxic megacolon. The colon was only slightly dilated and benign appearing. Importantly, HSV1 infection affected the postjunctional mechanisms of smooth muscle relaxation like the sildenafil-response proteins, which may have been responsible for defective nitrergic neurotransmission and the delayed transit. Orally administered polyethylene glycol reversed the gastrointestinal "obstruction," suggesting a mild functional type of slowed luminal transit, resembling constipation, rather than toxic megacolon, which cannot be reversed by an osmotic laxative without perforating the gut. The authors suggest that the mice did not develop HSV1 encephalitis, the commonly known cause of mortality. The premature death of some of the mice could be related to the bladder outlet obstruction, whose backflow effects may alter renal function, electrolyte abnormalities and death. Muscle strip recordings of mechanical relaxation after electrical field stimulation of gastrointestinal, urinary bladder or cavernosal tissues shall help obtain objective quantitative evidence of whether HSV infection indeed cause pelvic multi-organ dysfunction and impairment of autonomic neurotransmission and postjunctional electromechanical relaxation mechanisms of these organs.

Infant Formula Feeding Increases Hepatic Cholesterol 7α Hydroxylase (CYP7A1) Expression and Fecal Bile Acid Loss in Neonatal Piglets.

Background: During the postnatal feeding period, formula-fed infants have higher cholesterol synthesis rates and lower circulating cholesterol concentrations than their breastfed counterparts. Although this disparity has been attributed to the uniformly low dietary cholesterol content of typical infant formulas, little is known of the underlying mechanisms associated with this altered cholesterol metabolism phenotype. Objective: We aimed to determine the molecular etiology of diet-associated changes in early-life cholesterol metabolism with the use of a postnatal piglet feeding model. Methods: Two-day-old male and female White-Dutch Landrace piglets were fed either sow milk (Sow group) or dairy-based (Milk group; Similac Advance powder) or soy-based (Soy group; Emfamil Prosobee Lipil powder) infant formulas until day 21. In addition to measuring serum cholesterol concentrations, hepatic and intestinal genes involved in enterohepatic circulation of cholesterol and bile acids were analyzed by real-time reverse-transcriptase polymerase chain reaction and Western blot. Bile acid concentrations were measured by liquid chromatography-mass spectrometry in serum, liver, and feces. Results: Compared with the Sow group, hepatic cholesterol 7α hydroxylase (CYP7A1) protein expression was 3-fold higher in the Milk group (P < 0.05) and expression was 10-fold higher in the Soy group compared with the Milk group (P < 0.05). Likewise, fecal bile acid concentrations were 3-fold higher in the Soy group compared with the Milk group (P < 0.05). Intestinal mRNA expression of fibroblast factor 19 (Fgf19) was reduced in the Milk and Soy groups, corresponding to 54% and 67% decreases compared with the Sow group. In the Soy group, small heterodimer protein (SHP) protein expression was 30% lower compared with the Sow group (P < 0.05). Conclusions: These results indicate that formula feeding leads to increased CYP7A1 protein expression and fecal bile acid loss in neonatal piglets, and this outcome is linked to reduced efficacy in inhibiting CYP7A1 expression through FGF19 and SHP transcriptional repression mechanisms.

STAT3 Inhibition by Microtubule-Targeted Drugs: Dual Molecular Effects of Chemotherapeutic Agents.

To improve the effectiveness of anti-cancer therapies, it is necessary to identify molecular targets that are essential to a tumor cell but dispensable in a normal cell. Increasing evidence indicates that the transcription factor STAT3, which regulates the expression of genes controlling proliferation, survival, and self-renewal, constitutes such a target. Recently it has been found that STAT3 can associate with the cytoskeleton. Since many of the tumors in which STAT3 is activated, such as breast cancer and ovarian cancer, are responsive to drugs that target microtubules, we examined the effect of these compounds on STAT3. We found that microtubule stabilizers, such as paclitaxel, or microtubule inhibitors, such as vinorelbine, decrease the activating tyrosine phosphorylation of STAT3 in tumor cells and inhibit the expression of STAT3 target genes. Paclitaxel decreases the association between STAT3 and microtubules, and appears to decrease STAT3 phosphorylation through induction of a negative feedback regulator. The cytotoxic activity of paclitaxel in breast cancer cell lines correlates with its ability to decrease STAT3 phosphorylation. However, consistent with the necessity for expression of a negative regulator, treatment of resistant MDA-MB-231 cells with the DNA demethylating agent 5-azacytidine restores the ability of paclitaxel to block STAT3-dependent gene expression. Finally, the combination of paclitaxel and agents that directly target STAT3 has beneficial effects in killing STAT3-dependent cell lines. Thus, microtubule-targeted agents may exert some of their effects by inhibiting STAT3, and understanding this interaction may be important for optimizing rational targeted cancer therapies.

Microtubule-targeted chemotherapeutic agents inhibit signal transducer and activator of transcription 3 (STAT3) signaling.

The transcription factor signal transducer and activator of transcription 3 (STAT3) is inappropriately activated in the majority of breast tumors, especially in aggressive and invasive ones. In addition to driving the expression of genes promoting malignancy, STAT3 associates with tubulin and can promote cell migration. Because microtubule-targeted drugs are among the most active agents used in the treatment of breast cancer, we examined whether microtubule-based chemotherapy modulates STAT3 activity. When treated with paclitaxel or vinorelbine, breast cancer cells with constitutive activation of STAT3 display a loss of STAT3 phosphorylation, and paclitaxel disrupts the interaction of STAT3 with tubulin. Paclitaxel also inhibits cytokine-induced STAT3 activation. This effect is specific for microtubule-targeted agents, because other chemotherapeutic drugs, such as doxorubicin, have no effect on STAT3. The loss of STAT3 tyrosine phosphorylation is also reflected in an inhibition of expression of STAT3 target genes. This effect is not restricted to breast cancer, because similar effects are also seen in ovarian cancer and prostate cancer cells. Thus, in addition to their role in disrupting microtubule function, microtubule-targeted agents also suppress STAT3 signaling. This may be an important component of their activity, raising the possibility that microtubule targeted therapy may be particularly effective in tumors characterized by STAT3 activation.

Reciprocal effects of STAT5 and STAT3 in breast cancer.

Breast cancer is often associated with inappropriate activation of transcription factors involved in normal mammary development. Two related transcription factors, signal transducer and activator of transcription (STAT) 5 and STAT3, play important and distinct roles in mammary development and both can be activated in breast cancer. However, the relative contribution of these STATs to mammary tumorigenesis is unknown. We have found that primary human breast tumors displaying activation of both STATs are more differentiated than those with STAT3 activation alone and display more favorable prognostic characteristics. To understand this difference, we have analyzed the effect of these STATs on gene regulation and phenotype of mammary carcinoma cells. STAT5 and STAT3 mediate opposing effects on several key target genes, with STAT5 exerting a dominant role. Using a model system of paired breast cancer cell lines, we found that coactivation of STAT5 and STAT3 leads to decreased proliferation and increased sensitivity to the chemotherapeutic drugs paclitaxel and vinorelbine compared with cells that have only STAT3 activation. Thus, STAT5 can modify the effects of STAT3 from the level of gene expression to cellular phenotype and analysis of the activation state of both STAT5 and STAT3 may provide important diagnostic and prognostic information in breast cancer.