PDF file - 91K, List of cancer-associated genes containing potential Z-DNA elements

PDF file - 126K, H&E staining of canine breast cancer tissues and western blot analysis of ADAM-12 in canine tissues

Increased level of an inflammation-responsive transcription factor called serum amyloid A-activating factor (SAF-1) has been linked to the pathogenesis in human breast cancer. SAF-1 is found to promote vascular endothelial growth factor (VEGF) expression in breast carcinoma cells and boost angiogenesis. In an effort to develop a cellular mechanism to control VEGF expression, we sought to limit SAF-1 activity in breast cancer cells. We report here several targets within the SAF-1 mRNA for binding of microRNA-125b (miR-125b) and we show that VEGF expression is reduced in breast cancer cells when SAF-1 level is reduced with the microRNA action. Within the 3' un-translated region (UTR) of SAF-1 transcript, we have identified four highly conserved miR-125b responsive elements. We show that these miR-125b binding sites mediate repression of SAF-1 by miR-125b. Ectopic expression of miR-125b in nonmetastatic and metastatic breast cancer cells repressed SAF-1-mediated activity on VEGF promoter function and inhibited cancer cell migration and invasion potentials in vitro. Together, these results suggest that termination of SAF-1 function by miR-125b could be developed as a potential anti-VEGF and anti-angiogenic agent, which has high clinical relevance.

Besides the involvement of mTOR activity in several cancer conditions, evidence exists that increased total mTOR protein level might be linked to some cancer conditions such as colorectal carcinoma. The increase in total mTOR protein level in colon cancer was found to be associated with enhanced tumor progression and poor prognosis. Total mTOR protein level is elevated in breast cancer cells compared to their nonmalignant counterparts. High mTOR protein level in breast cancer cells could be attributed to decreased mTOR protein degradation, increased mTOR protein expression, or both. Increased protein expression may involve an increase in the gene expression. Here, we investigated the possibility of increased MTOR gene expression as a potential underlying cause of the elevated total mTOR protein in breast cancer cells. Our results suggest that transcription of MTOR gene is increased in the estrogen receptor positive (ER+) MCF-7 breast cancer cells compared to other breast cell lines. DNA sequencing of the MTOR promoter identified sequence variations in MCF-7 cells, which could be potentially involved in upregulation of mTOR expression. Among these variations is a truncation of guanine thymine dinucleotide (GT) n repeat region in MCF-7 cells, which might be possibly implicated in the elevated transcription of MTOR gene in these cells. Moreover, our results revealed that metformin treatment, profoundly decreased mTOR mRNA levels in MCF-7 breast cancer cells. In conclusion, unraveling the potential mechanisms involved in the regulation of mTOR expression in breast cancer cells could provide an avenue for optimizing the efficacy of breast cancer treatment regimens.

Activation of mTOR is implicated in the development and progression of breast cancer. mTOR inhibition exhibited promising antitumor effects in breast cancer; however, its effect is compromised by several feedback mechanisms. One of such mechanisms is the upregulation of mTOR pathway in breast cancer cells. Despite the established role of mTOR activation in breast cancer, the status of total mTOR protein and its impact on the tumor behavior and response to treatment are poorly understood. Besides, the mechanisms underlying mTOR protein degradation in normal and cancer breast cells are still largely unknown. We and others found that total mTOR protein level is elevated in breast cancer cells compared to their nonmalignant counterparts. We have detected defective proteolysis of mTOR protein in breast cancer cells, which could, at least in part, explain the high level of mTOR protein in these cells. We show that metformin treatment in MCF-7 breast cancer cells induced degradation of mTOR and sequestration of this protein in a perinuclear region. The decrease in mTOR protein level in these cells correlated positively with a concomitant inhibition of proliferation and migration potentials of these cells. These findings provided a novel mechanism for the metformin action in breast cancer treatment. Understanding the proteolytic mechanism responsible for mTOR level in breast cancer may pave the way for improving the efficacy of breast cancer treatment regimens and mitigating drug resistance as well as providing a basis for potential novel therapeutic modalities for breast cancer.

Abstract Tumor microenvironment (TME) plays a critical role in tumor growth, invasion and metastasis. In TME, epidermal growth factor receptor (EGFR) family members, including HER1, HER2, HER3 and HER4, are involved in determining aggressive growth of breast cancer due to their ability to transduce the growth promoting functions of growth factors. This activity is potentiated by the over-expression of these receptor molecules in cancer cells. To reduce the activity of EGFR molecules, various inhibitors have been developed. EGFR/HER1 tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, show antitumor activity but these drugs have not meet their primary goal of improved survival in the overall patient population. A similar TKI, lapatinib, has shown some limited success in breast cancer. While these therapeutic options focus on reducing the functional activity of EGFR protein, the fundamental and major problem of over-expression of EGFR in cancer cells has not been addressed. In two-thirds of aggressive breast cancer patients, transcriptional induction of EGFR causes high EGFR/HER1 level. Therefore, the goal of this study is to identify a molecular mechanism by which EGFR is over-expressed in breast cancer cells. To understand how transcriptional induction might occur, we have explored a novel biosynthetic pathway for EGFR over-expression. Our findings reveal that EGFR promoter is significantly more active in MDA-MB-231 cells in comparison to MDA-MB-468 cells. When transcription factor SAF-1 was ectopically expressed in these cells, EGFR promoter activity was further increased in MDA-MB-231 cells. Since MDA-MB-231 cells contain a highly active form of Ras, the data suggested a possible Ras-mediated activation of SAF-1 which in turn induces EGFR expression. Consistent with these findings, inhibition of K- and H-Ras, by using both siRNA and CRISPR/Cas9-mediated knock-out systems, reduced the expression of EGFR in MDA-MB-231 cells. The effect was more profound when K-Ras was targeted for inhibition. Since SAF-1/MAZ is seen to be activated by Ras, our data implicates K-Ras - SAF-1/MAZ - EGFR axis in breast cancer cell growth. Our findings may provide new targets for breast cancer therapy. Supported by grants from College of Veterinary Medicine Faculty Research Award Grant and MU Center for Botanical Interaction Studies Pilot Project Grant. Citation Format: Alpana Ray, Brett Havis, Bimal Ray. A novel regulatory mechanism involving Ras-mediated activation of the zinc-finger transcription factor, SAF-1/MAZ induces EGFR/HER1 expression in breast cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A65.

In the majority of breast cancers, overexpression and hyperactivation of Ras in the tumor microenvironment play significant role in promoting cancer cell growth, angiogenesis, and metastasis. We have previously shown that vascular endothelial growth factor (VEGF) expression in triple negative breast cancer cells is regulated, at least in part, by SAF-1 (serum amyloid A activating factor 1) transcription factor. In this study we show that transformation of normal MCF-10A breast epithelial cells by constitutively active, oncogenic Ras, induces the DNA-binding activity and transcription function of SAF-1. Furthermore, we show that inhibition of MEK/MAPK-signaling pathway prevents Ras-mediated activation of SAF-1. Interestingly, silencing of SAF-1 expression in breast cancer cells by SAF-1-specific short hairpin RNAs (shRNAs) significantly reduced H-Ras and K-Ras mRNA level. We show that SAF-1 is a direct transcriptional regulator of H-Ras and K-Ras and overexpression of SAF-1 increases H-Ras and K-Ras gene expression. Chromatin immunoprecipitation (ChIP) analyses demonstrated in vivo interaction of SAF-1 at highly purine-rich sequences present at the proximal promoter region, upstream of the transcription start site, in H-Ras and K-Ras genes. Previous studies have shown that these sequences are nuclease hypersensitive and capable of forming G4 quadruplex structure. Together, our results show the presence of a novel transactivating loop, in which, Ras and SAF-1 are interconnected. These findings will help defining molecular mechanisms of abnormal overexpression of Ras in breast tumors, which seldom show genetic Ras mutations.

The primary function of insulin is viewed as a hormone that controls blood glucose level. However, there is growing evidence that aberrant insulin level and insulin-mediated signaling can lead to cancer development and progression. The insulin-cancer relationship has stemmed from various observational and epidemiological studies, which linked higher incidence of cancer with central obesity, type II diabetes and other conditions associated with increased levels of circulating insulin, insulin resistance and hyperinsulinemic states. Increased risk of developing a range of cancers is also seen with a certain treatment options used to lower blood glucose level in diabetic patients. While metformin monotherapy has the lowest risk of developing cancer, in comparison, treatment with insulin or insulin secretagogues shows more likelihood to develop solid cancers. Cellular signaling initiated by insulin provides a clue regarding these diverse cellular outcomes. This review discusses how the insulin enacts such diverse physiological effects and the insulin-cancer relationship, with focus on the role of insulin signaling in cancer.

Abstract There is substantial evidence that metformin, an anti-diabetic biguanide, correlates with improved breast cancer response to chemotherapy. The exact mechanism of metformin's beneficial effect in breast cancer is yet to be fully understood. Metformin inhibits a serine/ threonine kinase called mammalian target of rapamycin (mTOR), which is a central regulator of various intracellular and extracellular stimuli. mTOR transduces signaling from growth factors, such as insulin, to stimulate mRNA translation. Insulin activates mTOR through PI3K-Akt-mediated phosphorylation and consequent activation of protein machinery to promote protein synthesis. Increased protein synthesis in cancer cells contributes to cancer progression. Improved response of breast cancer cells response to chemotherapy following metformin treatment suggests inhibition of mTOR in breast cancer regression. Being a serine / threonine kinase, mTOR may also have a potential role in the regulation of gene transcription via modulating the activity of some transcription factors, particularly, those involved in cell proliferation and differentiation. Kruppel-like factor 4 (KLF-4) is one such transcription factor which is involved in epithelial cells differentiation and proliferation and it is potentially regulated by mTOR. The objective of this study, therefore, was to investigate whether mTOR regulates gene expression by modulating the activity of KLF-4 in breast cancer. Our hypothesis was that mTOR inhibition by metformin results in the upregulation of KLF-4, which functions as a tumor suppressor. Elucidation of the molecular basis of metformin's effect on KLF-4 could provide the basis for adjuvant therapy in breast cancer. Western blot (WB) studies indicate overexpression of mTOR in some breast cancer cells compared to normal breast epithelial cells. In contrast, WB and Immunohistochemistry studies indicate overexpression of KLF-4 in normal breast cells compared to cancer breast cells. Metformin treatment, with and without insulin, resulted in decreased mTOR level with a concomitant increase in KLF-4 level in breast cancer cells. These results suggest a role of metformin in mediating the inverse relationship between mTOR and KLF-4 in breast cancer. Citation Format: Mohamed Alalem, Alpana Ray, Bimal Ray. Role of mTOR as a transcriptional regulator in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A042.

Abstract A disintegrin and metalloprotease domain-containing protein 12 (ADAM-12) is upregulated in many human cancers and promotes cancer metastasis. Increased urinary level of ADAM-12 in breast and bladder cancers correlates with disease progression. However, the mechanism of its induction in cancer remains less understood. Previously, we reported a Z-DNA–forming negative regulatory element (NRE) in ADAM-12 that functions as a transcriptional suppressor to maintain a low-level expression of ADAM-12 in most normal cells. We now report here that overexpression of ADAM-12 in triple-negative MDA-MB-231 breast cancer cells and breast cancer tumors is likely due to a marked loss of this Z-DNA–mediated transcriptional suppression function. We show that Z-DNA suppressor operates by interaction with methyl-CpG-binding protein, MeCP2, a prominent epigenetic regulator, and two members of the nuclear factor 1 family of transcription factors, NF1C and NF1X. While this tripartite interaction is highly prevalent in normal breast epithelial cells, both in vitro and in vivo, it is significantly lower in breast cancer cells. Western blot analysis has revealed significant differences in the levels of these 3 proteins between normal mammary epithelial and breast cancer cells. Furthermore, we show, by NRE mutation analysis, that interaction of these proteins with the NRE is necessary for effective suppressor function. Our findings unveil a new epigenetic regulatory process in which Z-DNA/MeCP2/NF1 interaction leads to transcriptional suppression, loss of which results in ADAM-12 overexpression in breast cancer cells. Cancer Res; 73(2); 736–44. ©2012 AACR.

Vascular endothelial growth factor (VEGF) is recognized as an important angiogenic factor that promotes angiogenesis in a series of pathological conditions, including cancer, inflammation, and ischemic disorders. We have recently shown that the inflammatory transcription factor SAF-1 is, at least in part, responsible for the marked increase of VEGF levels in breast cancer. Here, we show that SAF-1-mediated induction of VEGF is repressed by KLF-4 transcription factor. KLF-4 is abundantly present in normal breast epithelial cells, but its level is considerably reduced in breast cancer cells and clinical cancer tissues. In the human VEGF promoter, SAF-1- and KLF-4-binding elements are overlapping, whereas SAF-1 induces and KLF-4 suppresses VEGF expression. Ectopic overexpression of KLF-4 and RNAi-mediated inhibition of endogenous KLF-4 supported the role of KLF-4 as a transcriptional repressor of VEGF and an inhibitor of angiogenesis in breast cancer cells. We show that KLF-4 recruits histone deacetylases (HDACs) -2 and -3 at the VEGF promoter. Chronological ChIP assays demonstrated the occupancy of KLF-4, HDAC2, and HDAC3 in the VEGF promoter in normal MCF-10A cells but not in MDA-MB-231 cancer cells. Co-transfection of KLF-4 and HDAC expression plasmids in breast cancer cells results in synergistic repression of VEGF expression and inhibition of angiogenic potential of these carcinoma cells. Together these results identify a new mechanism of VEGF up-regulation in cancer that involves concomitant loss of KLF-4-HDAC-mediated transcriptional repression and active recruitment of SAF-1-mediated transcriptional activation.

Many studies have tested the consumption of foods and supplements to reduce exercise-induced muscle damage, but fasting itself is also worthy of investigation due to reports of beneficial effects of caloric restriction and/or intermittent fasting on inflammation and oxidative stress. This preliminary investigation compared indicators of exercise-induced muscle damage between upper-body untrained participants (N=29, 22yrs old (SD=3.34), 12 women) who completed 8h water-only fasts or ate a controlled diet in the 8h prior to five consecutive laboratory sessions. All sessions were conducted in the afternoon hours (i.e., post meridiem) and the women completed the first session while in the follicular phase of their menstrual cycles. Measures of muscle pain, resting elbow extension, upper arm girth, isometric strength, myoglobin (Mb), total nitric oxide (NO), interleukin 1beta (IL1b), and tumor necrosis factor alpha (TNFa) were collected before and after eccentric contractions of the non-dominant elbow flexors were completed. The fasting group's loss of elbow extension was less than the post-prandial group (p<.05, eta(2)=.10), but the groups did not change differently across time for any other outcome measures. However, significantly higher NO (p<.05, eta(2)=.22) and lower TNFa (p<.001, eta(2)=.53) were detected in the fasting group than the post-prandial group regardless of time. These results suggest intermittent fasting does not robustly inhibit the signs and symptoms of exercise-induced muscle damage, but such fasting may generally affect common indirect markers of muscle damage.

Abstract Angiogenesis plays an important role in the tumor growth and metastasis by facilitating increased blood supply and thus fueling unwanted growth factors and nutrients to the growing tumor. Vascular endothelial growth factor (VEGF) is recognized as a multifunctional angiogenic stimulator that stimulates blood vessel formation and endothelial cell survival. Recent therapeutic trials with anti-VEGF antibody bevacizumab (Avastin), although produced beneficial effects on some cancers including colorectal and lung cancers, had very limited beneficial outcome in breast cancer. The diverse response of avastin raised questions about the efficacy of anti-VEGF antibody approach for breast cancer therapy. Despite the disappointing outcome, it cannot be ignored that regulation of angiogenesis plays a critical role in breast cancer. We hypothesized that angiogenesis may be better controlled by blocking the synthesis of VEGF protein as opposed to the present approach that attempts to block the functions of VEGF by using antibodies. Induction of VEGF by a plethora of physiological and environmental stimuli strongly suggests the presence of multiple routes of VEGF synthesis, which are incompletely understood. We present evidence for a novel mode of transcriptional induction of VEGF, regulated by an inflammation-responsive transcription factor, SAF-1. Incidentally, inflammatory breast cancer exhibits increased angiogenesis and has a higher metastatic potential than noninflammatory breast cancer, supporting the notion that chronic inflammation plays a strong role in angiogenesis and in the progression of cancer. SAF-1 is present in high abundance in human breast cancer tissues. We show that inhibition of endogenous SAF-1 by antisense shRNA can markedly inhibit VEGF expression and block cancer cell-supported vascular endothelial cell function and angiogenesis. Furthermore, SAF-1 inhibition by shRNA reduces tumor growth in mouse tumor model. We provide evidence that SAF-1-mediated induction of VEGF remains suppressed by KLF-4 transcription factor which is abundantly present in normal breast epithelial cells but absent in breast cancer cells. Mutually exclusive interaction of KLF-4 and SAF-1 at the VEGF promoter provides a mechanism for regulation of low-expression of VEGF in normal breast tissues. In correlation, lack of KLF-4 in breast cancer cells appears to be linked to un-restricted interaction of SAF-1 leading to the induction of VEGF expression. Together, our studies provide a new insight into the molecular mechanisms of VEGF expression regulated by SAF-1 and its impact in angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5278. doi:1538-7445.AM2012-5278

Angiogenesis plays a significant role in cancer by providing increased blood supply to the affected tissues and thus bringing in growth factors, cytokines, and various nutrients for tumor growth. VEGF is the most prominent angiogenic agent that is markedly induced in cancer. Induction of VEGF has been widely studied but as cancer cells are quite adept at acquiring new alternative processes to circumvent surrounding environmental pressures, our understanding of the molecular mechanisms regulating VEGF expression in cancer, especially in triple-negative breast cancer cells, remains incomplete. Here, we present evidence of a novel mode of VEGF induction in triple-negative MDA-MB-231 breast cancer cells that is regulated by serum amyloid A activating factor 1 (SAF-1) transcription factor. Inhibition of SAF-1 by antisense short hairpin RNA profoundly reduces VEGF expression along with reduction in endothelial cell proliferation and migration. By both in vitro and in vivo molecular studies, we show that the effect of SAF-1 is mediated through its direct interaction with the VEGF promoter. In correlation, DNA-binding activity of SAF-1 is found to be significantly higher in MDA-MB-231 breast cancer cells. Examination of several breast cancer samples further revealed that SAF-1 is overexpressed in clinical breast cancer tissues. Taken together, these findings reveal that SAF-1 is a hitherto unrecognized participant in inducing VEGF expression in triple-negative breast cancer cells, an aggressive form of breast cancer that currently lacks effective treatment options. Suppression of SAF-1 activity in these cells can inhibit VEGF expression, providing a possible new method to control angiogenesis.

Upregulation of ADAM-12, a novel member of the multifunctional ADAM family of proteins is linked to cancer, arthritis and cardiac hypertrophy. Basal expression of ADAM-12 is very low in adult tissues but rises markedly in response to certain physiological cues, such as during pregnancy in the placenta, during development in neonatal skeletal muscle and bone and in regenerating muscle. Studies on ADAM-12 regulation have identified a highly conserved negative regulatory element (NRE) at the 5′-UTR of human ADAM-12 gene, which acts as a transcriptional repressor. The NRE contains a stretch of dinucleotide-repeat sequence that is able to adopt a Z-DNA conformation both in vitro and in vivo and interacts with hZαADAR1, a bona fide Z-DNA-binding protein. Substitution of the dinucleotide-repeat-element with a non-Z-DNA-forming sequence inhibited NRE function. We have detected a NRE DNA-binding protein activity in several tissues where ADAM-12 expression is low while no such activity was seen in the placenta where ADAM-12 expression is high. These observations suggest that interaction of these proteins with ADAM-12 NRE is critical for transcriptional repression of ADAM-12. We also show that the Z-DNA forming transcriptional repressor element, by interacting with these putative Z-DNA-binding proteins, is involved in the maintenance of constitutive low-level expression of human ADAM-12. Together these results provide a foundation for therapeutic down-regulation of ADAM-12 in cancer, arthritis and cardiac hypertrophy.

Animal studies have supported the immunological benefits of caloric restriction, but clinical trials of such diets in humans are scarce. Regardless, several studies in humans have shown differences in fasting and postprandial levels of inflammatory markers, which may be relevant to studies of exercise-induced muscle damage. PURPOSE: This preliminary investigation compared the signs and symptoms of exercise-induced muscle damage between participants who completed their laboratory sessions in a fasting or postprandial state. METHODS: Upper-body untrained participants (N = 29, 22 yrs old (SD = 3.34), 41.4% women) visited the lab in p.m. hours for five consecutive days after having either fasted for the previous 8 hours or eaten only a supplied meal within the previous 4-5 hours. (The supplied meal consisted of 810-860c, 250-320 fat c, 28-35g fat, 10-13g sat fat, 40-45mg cholesterol, 6-7g fiber, 101-104g carbohydrate, and 32-34g protein.) Measures of muscle pain, resting elbow extension, upper arm girth, isometric strength, myoglobin (Mb), total nitric oxide (NO), interleukin 1beta (IL1b), and tumor necrosis factor (TNFalpha) were collected before and after eccentric contractions of the non-dominant elbow flexors were completed. RESULTS: The loss of elbow extension peaked significantly more slowly for the fasting group than the postprandial group (F3, 72 = 3.01, p <.05, eta2 =.10), but the groups did not change differently across time for any other outcome measures. However, significantly lower strength (53.4+3.7 vs. 73.1+5.2% body weight, p <.05), higher NO (27.06+1.37 vs. 20.73+1.88 μmole/L, p <.05), and lower TNFalpha (2.18+0.18 vs. 3.89+0.25 pg/mL, p <.05), were detected in the fasting group than the postprandial group regardless of time. CONCLUSIONS: These results suggest that eating behavior within 8 hours of lab visits may be a meaningful source of variability in human exercise-induced muscle damage protocols, but intermittent fasting does not generally inhibit the signs and symptoms of exercise-induced muscle damage. Additional research on potential protective effects of caloric restriction (i.e., short-term fasting) on the exercise-induced muscle damage model in humans is warranted. Supported by NIAMS (KO1 AR050146) to E. A. Dannecker, PhD, ATC.