Comparison of tumor growth assessment using GFP fluorescence and DiI labeling in a zebrafish xenograft model.

DiI is a lipophilic fluorescent dye frequently used to label and trace cells in cell cultures and xenograft models. However, DiI can also transfer from labeled to unlabeled cells, including host organism cells, and label dead cells obscuring interpretation of the results. These limitations of DiI labeling in xenograft models have not been thoroughly investigated. Here we labeled green fluorescent protein (GFP)-expressing MDA-MB-231 cells with DiI to directly compare tumor growth assessment in zebrafish xenografts using the DiI labeling and GFP fluorescence. Our results indicate that the DiI based assessment significantly overestimated tumor growth in zebrafish xenograft models compared to the GFP fluorescence based assessment. The imaging of DiI labeled GFP-expressing MDA-MB-231 cell cultures indicated that the DiI labeling of the membrane is uneven. Analysis of the DiI labeled GFP-expressing MDA-MB-231 cell cultures with flow cytometry indicated that the DiI labeling varied over time while the GFP fluorescence remained unchanged, suggesting that the GFP fluorescence is a more reliable signal for monitoring tumor progression than the DiI labeling. Taken together, our results demonstrate limitations of using DiI labeling for xenograft models and emphasize the need for validating the results based on DiI labeling with other orthogonal methods, such as the ones utilizing genetically encoded fluorophores.

Loss of RARRES1 function Promotes Follicular Lymphomagenesis and Inhibits B cell Differentiation in Mice.

Retinoic acid receptor responder 1 (RARRES1) is among the most commonly methylated loci in multiple cancers. RARRES1 regulates mitochondrial and fatty acid metabolism, stem cell differentiation, and survival of immortalized cell lines in vitro. Here, we created constitutive Rarres1 knockout (Rarres1-/-) mouse models to study RARRES1 function in vivo. Rarres1-/- embryonic fibroblasts regulated tubulin glutamylation, cell metabolism, and survival, recapitulating RARRES1 function in immortalized cell lines. In two mouse strains, loss of Rarres1 led to a markedly increased dose-dependent incidence of follicular lymphoma (FL). Prior to lymphoma formation, Rarres1-/- B cells have compromised activation, maturation, differentiation into antibody-secreting plasma cells, and cell cycle progression. Rarres1 ablation increased B cell survival and led to activation of the unfolded protein response (UPR) and heat shock response (HSR). Rarres1 deficiency had differential effects on cellular metabolism, with increased bioenergetic capacity in fibroblasts, and minor effects on bioenergetics and metabolism in B cells. These findings reveal that RARRES1 is a bona fide tumor suppressor in vivo and the deletion in mice promotes cell survival, and reduces B cell differentiation with B cell autonomous and non-autonomous functions.

Monoclonal Antibodies for the Detection of a Specific Cyclic DNA Adduct Derived from ω-6 Polyunsaturated Fatty Acids.

Lipid peroxidation of polyunsaturated fatty acids (PUFAs) is an endogenous source of α,ß-unsaturated aldehydes that react with DNA producing a variety of cyclic adducts. The mutagenic cyclic adducts, specifically those derived from oxidation of ω-6 PUFAs, may contribute to the cancer promoting activities associated with ω-6 PUFAs. ( E)-4-Hydroxy-2-nonenal (HNE) is a unique product of ω-6 PUFAs oxidation. HNE reacts with deoxyguanosine (dG) yielding mutagenic 1, N2-propanodeoxyguanosine adducts (HNE-dG). Earlier studies showed HNE can also be oxidized to its epoxide (EH), and EH can react with deoxyadenosine (dA) forming the well-studied εdA and the substituted etheno adducts. Using a liquid chromatography-based tandem mass spectroscopic (LC-MS/MS) method, we previously reported the detection of EH-derived 7-(1',2'-dihydroxyheptyl)-1, N6-ethenodeoxyadenosine (DHHεdA) as a novel endogenous background adduct in DNA from rodent and human tissues. The formation, repair, and mutagenicity of DHHεdA and its biological consequences in cells have not been investigated. To understand the roles of DHHεdA in carcinogenesis, it is important to develop an immuno-based assay to detect DHHεdA in cells and tissues. In this study we describe the development of monoclonal antibodies specifically against DHHεdA and its application to detect DHHεdA in human cells.

Discovery of a stem-like multipotent cell fate.

Adipose derived stem cells (ASCs) can be obtained from lipoaspirates and induced in vitro to differentiate into bone, cartilage, and fat. Using this powerful model system we show that after in vitro adipose differentiation a population of cells retain stem-like qualities including multipotency. They are lipid (-), retain the ability to propagate, express two known stem cell markers, and maintain the capacity for trilineage differentiation into chondrocytes, adipocytes, and osteoblasts. However, these cells are not traditional stem cells because gene expression analysis showed an overall expression profile similar to that of adipocytes. In addition to broadening our understanding of cellular multipotency, our work may be particularly relevant to obesity-associated metabolic disorders. The adipose expandability hypothesis proposes that inability to differentiate new adipocytes is a primary cause of metabolic syndrome in obesity, including diabetes and cardiovascular disease. Here we have defined a differentiation-resistant stem-like multipotent cell population that may be involved in regulation of adipose expandability in vivo and may therefore play key roles in the comorbidities of obesity.

Ly6E/K Signaling to TGFß Promotes Breast Cancer Progression, Immune Escape, and Drug Resistance.

Stem cell antigen Sca-1 is implicated in murine cancer stem cell biology and breast cancer models, but the role of its human homologs Ly6K and Ly6E in breast cancer are not established. Here we report increased expression of Ly6K/E in human breast cancer specimens correlates with poor overall survival, with an additional specific role for Ly6E in poor therapeutic outcomes. Increased expression of Ly6K/E also correlated with increased expression of the immune checkpoint molecules PDL1 and CTLA4, increased tumor-infiltrating T regulatory cells, and decreased natural killer (NK) cell activation. Mechanistically, Ly6K/E was required for TGFß signaling and proliferation in breast cancer cells, where they contributed to phosphorylation of Smad1/5 and Smad2/3. Furthermore, Ly6K/E promoted cytokine-induced PDL1 expression and activation and binding of NK cells to cancer cells. Finally, we found that Ly6K/E promoted drug resistance and facilitated immune escape in this setting. Overall, our results establish a pivotal role for a Ly6K/E signaling axis involving TGFß in breast cancer pathophysiology and drug response, and highlight this signaling axis as a compelling realm for therapeutic invention. Cancer Res; 76(11); 3376-86. ©2016 AACR.

Nucleotide excision repair deficiency increases levels of acrolein-derived cyclic DNA adduct and sensitizes cells to apoptosis induced by docosahexaenoic acid and acrolein.

The acrolein derived cyclic 1,N(2)-propanodeoxyguanosine adduct (Acr-dG), formed primarily from ω-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) under oxidative conditions, while proven to be mutagenic, is potentially involved in DHA-induced apoptosis. The latter may contribute to the chemopreventive effects of DHA. Previous studies have shown that the levels of Acr-dG are correlated with apoptosis induction in HT29 cells treated with DHA. Because Acr-dG is shown to be repaired by the nucleotide excision repair (NER) pathway, to further investigate the role of Acr-dG in apoptosis, in this study, NER-deficient XPA and its isogenic NER-proficient XAN1 cells were treated with DHA. The Acr-dG levels and apoptosis were sharply increased in XPA cells, but not in XAN1 cells when treated with 125µM of DHA. Because DHA can induce formation of various DNA damage, to specifically investigate the role of Acr-dG in apoptosis induction, we treated XPA knockdown HCT116+ch3 cells with acrolein. The levels of both Acr-dG and apoptosis induction increased significantly in the XPA knockdown cells. These results clearly demonstrate that NER deficiency induces higher levels of Acr-dG in cells treated with DHA or acrolein and sensitizes cells to undergo apoptosis in a correlative manner. Collectively, these results support that Acr-dG, a ubiquitously formed mutagenic oxidative DNA adduct, plays a role in DHA-induced apoptosis and suggest that it could serve as a biomarker for the cancer preventive effects of DHA.

Inhibition of c-Src blocks oestrogen-induced apoptosis and restores oestrogen-stimulated growth in long-term oestrogen-deprived breast cancer cells.

PURPOSE: Our publications demonstrate that physiological concentrations of oestrogen (E2) induce endoplasmic reticulum and oxidative stress which finally result in apoptosis in E2-deprived breast cancer cells, MCF-7:5C. c-Src is involved in the process of E2-induced stress. To mimic the clinical administration of c-Src inhibitors, we treated cells with either E2, a c-Src inhibitor PP2, or the combination for 8 weeks to further explore the apoptotic potential of the c-Src inhibitor and E2 on MCF-7:5C cells. METHODS: Protein levels of receptors and signalling pathways were examined by immunoblotting. Expression of mRNA was detected through real-time polymerase chain reaction (PCR). Cell cycles were analysed by flow cytometry. RESULTS: Long-term treatment with PP2 alone or E2 alone decreased cell growth. In contrast, a combination of PP2 and E2 blocked apoptosis and the resulting cell line (MCF-7:PF) was unique, as they grew vigorously in culture with physiological levels of E2, which could be blocked by the pure antioestrogen ICI182,780. One major change was that PP2 collaborated with E2 to increase the level of insulin-like growth factor-1 receptor beta (IGF-1Rß). Blockade of IGF-1Rß completely abolished E2-stimulated growth in MCF-7:PF cells. Furthermore, combination treatment up-regulated transcription factors, Twist1 and Snail, and repressed E-cadherin expression which made MCF-7:PF cells display a characteristic phenotype of epithelial-mesenchymal transition (EMT). CONCLUSIONS: These data illustrate the role of the c-Src inhibitor to block E2-induced apoptosis and enhance E2-stimulated growth. Caution must be exercised when considering c-Src inhibitors in clinical trials following the development of acquired resistance to aromatase inhibitors, especially in the presence of the patient's own oestrogen.

c-Src modulates estrogen-induced stress and apoptosis in estrogen-deprived breast cancer cells.

The emergence of anti-estrogen resistance in breast cancer is an important clinical phenomenon affecting long-term survival in this disease. Identifying factors that convey cell survival in this setting may guide improvements in treatment. Estrogen (E2) can induce apoptosis in breast cancer cells that have been selected for survival after E2 deprivation for long periods (MCF-7:5C cells), but the mechanisms underlying E2-induced stress in this setting have not been elucidated. Here, we report that the c-Src kinase functions as a key adapter protein for the estrogen receptor (ER, ESR1) in its activation of stress responses induced by E2 in MCF-7:5C cells. E2 elevated phosphorylation of c-Src, which was blocked by 4-hydroxytamoxifen (4-OHT), suggesting that E2 activated c-Src through the ER. We found that E2 activated the sensors of the unfolded protein response (UPR), IRE1α (ERN1) and PERK kinase (EIF2AK3), the latter of which phosphorylates eukaryotic translation initiation factor-2α (eIF2α). E2 also dramatically increased reactive oxygen species production and upregulated expression of heme oxygenase HO-1 (HMOX1), an indicator of oxidative stress, along with the central energy sensor kinase AMPK (PRKAA2). Pharmacologic or RNA interference-mediated inhibition of c-Src abolished the phosphorylation of eIF2α and AMPK, blocked E2-induced ROS production, and inhibited E2-induced apoptosis. Together, our results establish that c-Src kinase mediates stresses generated by E2 in long-term E2-deprived cells that trigger apoptosis. This work offers a mechanistic rationale for a new approach in the treatment of endocrine-resistant breast cancer.

Detection of acrolein-derived cyclic DNA adducts in human cells by monoclonal antibodies.

Acrolein (Acr) is a ubiquitous environmental pollutant found in cigarette smoke and automobile exhaust. It can also be produced endogenously by oxidation of polyunsaturated fatty acids. The Acr-derived 1,N(2)-propanodeoxyguanosine (Acr-dG) adducts in DNA are mutagenic lesions that are potentially involved in human cancers. In this study, monoclonal antibodies were raised against Acr-dG adducts and characterized using ELISA. They showed strong reactivity and specificity toward Acr-dG, weaker reactivity toward crotonaldehyde- and trans-4-hydroxy-2-nonenal-derived 1,N(2)-propanodeoxyguanosines, and weak or no reactivity toward 1,N(6)-ethenodeoxyadenosine and 8-oxo-deoxyguanosine. Using these antibodies, we developed assays to detect Acr-dG in vivo: first, a simple and quick FACS-based assay for detecting these adducts directly in cells; second, a highly sensitive direct ELISA assay for measuring Acr-dG in cells and tissues using only 1 µg of DNA without DNA digestion and sample enrichment; and third, a competitive ELISA for better quantitative measurement of Acr-dG levels in DNA samples. The assays were validated using Acr-treated HT29 cell DNA samples or calf thymus DNA, and the results were confirmed by LC-MS/MS-MRM. An immunohistochemical assay was also developed to detect and visualize Acr-dG in HT29 cells as well as in human oral cells. These antibody-based methods provide useful tools for the studies of Acr-dG as a cancer biomarker and of the molecular mechanisms by which cells respond to Acr-dG as a ubiquitous DNA lesion.

Phase I/II study of sorafenib with anastrozole in patients with hormone receptor positive aromatase inhibitor resistant metastatic breast cancer.

We evaluated the use of sorafenib to overcome resistance to aromatase inhibitors (AIs) in patients with metastatic breast cancer who had disease recurrence or progression while on AIs. We performed a multi-institution phase I/II study of sorafenib and anastrozole 1 mg daily in 35 postmenopausal females with hormone receptor positive metastatic breast cancer resistant to AIs. Primary objectives were to determine the dose of sorafenib in conjunction with anastrozole and the clinical benefit rate (CBR) (complete response [CR], partial response [PR], or stable disease [SD] ≥ 24 weeks). Secondary objectives were to determine toxicity and to evaluate if response was associated with change in number of circulating endothelial cells or circulating endothelial progenitor cells. Based on the phase I portion, sorafenib 400 mg twice daily was selected as the phase II dose. Among 35 patients, 7 had SD ≥ 24 weeks, 1 had PR ≥ 24 weeks, and 14 had progressive disease (PD) ≤ 24 weeks, corresponding to a CBR of 23%. The most common adverse events (all; Grade 3/4) were fatigue (66%; 17%), diarrhea (63%; 6%), nausea (60%; 9%), and hand-foot syndrome (57%; 34%). Dose reduction occurred in 77% of the patients and 31% came off study due to toxicity. The combination of sorafenib and anastrozole demonstrated a 23% CBR in patients with hormone receptor positive, AI-resistant metastatic breast cancer, which may be attributable to the restoration of sensitivity to AIs. Toxicities occurred frequently resulting in a high rate of discontinuation.

Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells.

Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R.

Characterization of anti-islet cytotoxic human T-cell clones from patients with type 1 diabetes mellitus.

To identify important anti-islet T-cells and their target antigen(s), we have isolated and characterized seventeen human T-cell clones which are reactive to an extract of rat insulinoma (RIN) cells from three children with new onset type 1 diabetes mellitus (T1D). Of these 17 clones, 15 were found tissue specific. Six of eight tested tissue specific clones did not recognize known islet antigens such as GAD, 52 kDa islet protein, insulin, ICA512, and heat shock protein 60 (hsp60), suggesting that these clones recognize an autoantigen not previously identified. All tested clones were phenotypically CD4 and functionally Th0 or Th0/Th1 cells. One RIN extract reactive clone (2E9) recognized hsp60 and was CD4 and TCR alpha/beta positive. This clone also proliferated in response to human and rat islets suggesting that the antigen is conserved between species. This clone and 75% of all the tested RIN reactive clones exhibited anti-islet cytotoxicity by lysing target cells coated with RIN extract. HLA DR determinants may play a role in this cytotoxic activity since preincubation with HLA DR antibody decreased the anti-islet cytoxicity of the two tested clones. In conclusion, we have isolated RIN reactive CD4+T-cell clones from diabetic subjects, six of which appears tissue specific and non-reactive to putative important islet antigens, and in turn may be recognizing yet undiscovered islet antigens. The high frequency anti-islet cytotoxic properties of the islet reactive clones provides evidence for a role of CD4+ cytotoxic T-lymphocytes in the diabetic process. Further, the isolation of hsp60 reactive clone with anti-islet cytotoxic properties suggests that cell mediated immunity against hsp60 may be important in the pathogenesis of diabetes.

Tachyplesin activates the classic complement pathway to kill tumor cells.

Tachyplesin is a small, cationic peptide that possesses antitumor properties. However, little is known about its action mechanism. We used phage display to identify a protein that interacted with tachyplesin and isolated a sequence corresponding to the collagen-like domain of C1q, a key component in the complement pathway. Their interaction was subsequently confirmed by both ELISA and affinity precipitation. Tachyplesin seemed to activate the classic complement cascade because it triggered several downstream events, including the cleavage and deposition of C4 and C3 and the formation of C5b-9. When TSU tumor cells were treated with tachyplesin in the presence of serum, activated C4b and C3b could be detected on tumor cells by flow cytometry, Western blotting, and confocal microscopy. However, this effect was blocked when the tumor cells were treated with hyaluronidase or a large excess of hyaluronan, indicating that hyaluronan or related glycosaminoglycans were involved in this process. Treatment of cells with tachyplesin and serum increased in membrane permeability as indicated by the ability of FITC-dextran to enter the cytoplasm. Finally, the combination of tachyplesin and human serum markedly inhibited the proliferation and caused death of TSU cells, and these effects were attenuated if the serum was heat-inactivated or if hyaluronidase was added. Taken together, these observations suggest that tachyplesin binds to both hyaluronan on the cell surface and C1q in the serum and activates the classic complement cascade, which damages the integrity of the membranes of the tumor cells resulting in their death.

A peptide with three hyaluronan binding motifs inhibits tumor growth and induces apoptosis.

A number of hyaluronan (HA) binding proteins such as soluble CD44, receptor for hyaluronan-mediated motility (RHAMM), and metastatin inhibit tumor growth and metastasis. To determine whether the HA binding motif is the element responsible for the antitumor effect of this family of proteins, we examined the biological activity of a 42-amino acid peptide (designated as BH-P) that contains three HA binding motifs [B(X(7))B] from human brain HA binding protein. In initial experiments with cultured cells, we found that synthetic BH-P inhibited the proliferation and colony formation of tumor cells. It also blocked the growth of tumors on the chorioallantoic membranes of 10-day chicken embryos. In addition, MDA-435 melanoma cells that had been transfected with an expression vector for BH-P grew at a slower rate in nude mice than the vector-alone transfectants. Final studies revealed that the BH-P could activate caspase-8, caspase-3, and poly(ADP-ribose) polymerase and trigger the apoptosis of tumor cells. Taken together, these results suggest that the HA binding motif that is present in HA binding proteins may be responsible for the antitumor effect exerted by the members of this family.