Obesity and poor diet often go hand-in-hand, altering metabolic signaling and thereby impacting breast cancer risk and outcomes. We have recently demonstrated that dietary patterns modulate mammary microbiota populations. An important and largely open question is whether the microbiome of the gut and mammary gland mediates the dietary effects on breast cancer. To address this, we performed fecal transplants between mice on control or high-fat diets (HFD) and recorded mammary tumor outcomes in a chemical carcinogenesis model. HFD induced protumorigenic effects, which could be mimicked in animals fed a control diet by transplanting HFD-derived microbiota. Fecal transplants altered both the gut and mammary tumor microbiota populations, suggesting a link between the gut and breast microbiomes. HFD increased serum levels of bacterial lipopolysaccharide (LPS), and control diet–derived fecal transplant reduced LPS bioavailability in HFD-fed animals. In vitro models of the normal breast epithelium showed that LPS disrupts tight junctions (TJ) and compromises epithelial permeability. In mice, HFD or fecal transplant from animals on HFD reduced expression of TJ-associated genes in the gut and mammary gland. Furthermore, infecting breast cancer cells with an HFD-derived microbiome increased proliferation, implicating tumor-associated bacteria in cancer signaling. In a double-blind placebo-controlled clinical trial of patients with breast cancer administered fish oil supplements before primary tumor resection, dietary intervention modulated the microbiota in tumors and normal breast tissue. This study demonstrates a link between the gut and breast that mediates the effect of diet on cancer. Significance: This study demonstrates that diet shifts the microbiome in the gut and the breast tumor microenvironment to affect tumorigenesis, and oral dietary interventions can modulate the tumor microbiota in patients with breast cancer.

Angiotensin-(1–7) (Ang-(1–7)) is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic and pro-oxidant effects of the Ang II-AT1 receptor axis. We previously identified a peptidase activity from sheep brain, proximal tubules and human HK-2 proximal tubule cells that metabolized Ang-(1–7); thus, the present study isolated and identified the Ang-(1–7) peptidase. Utilizing ion exchange and hydrophobic interaction chromatography, a single 80 kDa protein band on SDS-PAGE was purified from HK-2 cells. The 80 kDa band was excised, the tryptic digest peptides analyzed by LC-MS and a protein was identified as the enzyme dipeptidyl peptidase 3 (DPP 3, EC: 3.4.14.4). A human DPP 3 antibody identified a single 80 kDa band in the purified enzyme preparation identical to recombinant human DPP 3. Both the purified Ang-(1–7) peptidase and DPP 3 exhibited an identical hydrolysis profile of Ang-(1–7) and both activities were abolished by the metallopeptidase inhibitor JMV-390. DPP 3 sequentially hydrolyzed Ang-(1–7) to Ang-(3–7) and rapidly converted Ang-(3–7) to Ang-(5–7). Kinetic analysis revealed that Ang-(3–7) was hydrolyzed at a greater rate than Ang-(1–7) [17.9 vs. 5.5 nmol/min/μg protein], and the Km for Ang-(3–7) was lower than Ang-(1–7) [3 vs. 12 μM]. Finally, chronic treatment of the HK-2 cells with 20 nM JMV-390 reduced intracellular DPP 3 activity and tended to augment the cellular levels of Ang-(1–7). We conclude that DPP 3 may influence the cellular expression of Ang-(1–7) and potentially reflect a therapeutic target to augment the actions of the peptide.

Angiotensin 1-7 [ANG-(1-7)] is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic, and pro-oxidant effects of ANG II. We previously identified an peptidase that preferentially metabolized ANG-(1-7) to ANG-(1-4) in the brain medulla and cerebrospinal fluid (CSF) of sheep (Marshall AC, Pirro NT, Rose JC, Diz DI, Chappell MC. J Neurochem 130: 313-323, 2014); thus the present study established the expression of the peptidase in the kidney. Utilizing a sensitive HPLC-based approach, we demonstrate a peptidase activity that hydrolyzed ANG-(1-7) to ANG-(1-4) in the sheep cortex, isolated tubules, and human HK-2 renal epithelial cells. The peptidase was markedly sensitive to the metallopeptidase inhibitor JMV-390; human HK-2 cells expressed subnanomolar sensitivity (IC50 = 0.5 nM) and the highest specific activity (123 ± 5 fmol·min(-1)·mg(-1)) compared with the tubules (96 ± 12 fmol·min(-1)·mg(-1)) and cortex (107 ± 9 fmol·min(-1)·mg(-1)). The peptidase was purified 41-fold from HK-2 cells; the activity was sensitive to JMV-390, the chelator o-phenanthroline, and the mercury-containing compound p-chloromercuribenzoic acid (PCMB), but not to selective inhibitors against neprilysin, neurolysin and thimet oligopeptidase. Both ANG-(1-7) and its endogenous analog [Ala(1)]-ANG-(1-7) (alamandine) were preferentially hydrolyzed by the peptidase compared with ANG II, [Asp(1)]-ANG II, ANG I, and ANG-(1-12). Although the ANG-(1-7) peptidase and insulin-degrading enzyme (IDE) share similar inhibitor characteristics of a metallothiolendopeptidase, we demonstrate marked differences in substrate specificity, which suggest these peptidases are distinct. We conclude that an ANG-(1-7) peptidase is expressed within the renal proximal tubule and may play a potential role in the renal renin-angiotensin system to regulate ANG-(1-7) tone.

The renal proximal tubules are a key functional component of the kidney and express the angiotensin precursor angiotensinogen; however, it is unclear the extent that tubular angiotensinogen reflects local synthesis or internalization. Therefore, the current study established the extent to which angiotensinogen is internalized by proximal tubules and the intracellular distribution. Proximal tubules were isolated from the kidney cortex of male sheep by enzymatic digestion and a discontinuous Percoll gradient. Tubules were incubated with radiolabeled I-angiotensinogen for 2 h at 37°C in serum/phenol-free DMEM/F12 media. Approximately 10% of exogenousI-angiotensinogen was internalized by sheep tubules. Subcellular fractionation revealed that 21 ± 4% of the internalized I-angiotensinogen associated with the mitochondrial fraction with additional labeling evident in the nucleus (60 ± 7%), endoplasmic reticulum (4 ± 0.5%), and cytosol (15 ± 4%; = 4). Subsequent studies determined whether mitochondria directly internalized I-angiotensinogen using isolated mitochondria from renal cortex and human HK-2 proximal tubule cells. Sheep cortical and HK-2 mitochondria internalizedI-angiotensinogen at a comparable rate of (33 ± 9 vs. 21 ± 10 pmol·min·mg protein; = 3). Lastly, unlabeled angiotensinogen (100 nM) competed forI-angiotensinogen uptake to a greater extent than human albumin in HK-2 mitochondria (60 ± 2 vs. 16 ± 13%; < 0.05, = 3). Collectively, our data demonstrate angiotensinogen import and subsequent trafficking to the mitochondria in proximal tubules. We conclude that this pathway may constitute a source of the angiotensinogen precursor for the mitochondrial expression of angiotensin peptides.