Endocrine Active Chemicals in Breast Cancer Cells: Environmental Impacts on Growth, Signaling and Epigenetic Pathways, and Drug Response

Individuals living in industrialized regions of Westernized societies are exposed to environmental contaminants by many routes, including plastics, personal care items and other consumer products. Endocrine-active chemicals (EACs), many of which are estrogenic, have been detected in human biofluids and breast tissue, warranting investigation of roles in mammary tumorigenesis. This dissertation explores the molecular changes that occur in breast cancer cells upon treatment with relevant human exposure levels of bisphenol-A (BPA), methylparaben (MP), propylparaben (PP), and decabromodiphenyl ether (DBDE), including mixtures thereof with or without 17β-estradiol (E2). Nanomolar (nM) concentrations of these EACs induced viability increases in MCF-7 (ERα+) similar to picomolar (pM) E2 concentrations, but EAC mixtures did not produce additive effects. MDA-MB-231 viability was unaltered by EACs in the absence of E2 but was significantly increased with exposure to five EACs combined with 50 pM E2. To examine ERα gene regulation, transactivation assays in ERα-transfected HepG2 cells confirmed that nanomolar BPA could induce ER-driven transcription, and suggested an additive effect of EAC mixtures. Non-genomic ERα functions were also investigated in breast cancer cells via high-throughput microscopy and quantitative immunofluorescence with the Hermes/WiScan System. MCF-7 cells revealed a 50% increase in phosphorylated (P)-ERα Serine 167 expression after 30-minute exposure to 100 nM PP, followed by alterations in nuclear/cytosolic localization after 24 hours. Signaling/epigenetic pathways were assessed with fifteen additional protein markers. At 100 nM, EACs induced expression of P-ERK, β-catenin, and epigenetic marks at H3K4me2, H3K9acetyl, and H3K27me2, while decreasing levels of AR and LSD1. Similarities and differences in proteomic expression patterns were observed between E2 and EACs. These pathways were further found to influence breast cancer progression, drug response and resistance; EACs were shown here to increase Tamoxifen IC50 in MCF-7 cells, but further sensitized them to Doxorubicin toxicity. A proteomic marker shown to undergo nuclear localization changes during observed EAC-altered Tamoxifen responses was P-ERα-S167. After extended low-level EAC exposures (60 days), a four-day EAC-withdrawal was sufficient to reset MCF-7 drug responses to control levels. These results reveal that EACs have diverse cellular mechanisms involving genomic and proteomic ERα effects and signaling/epigenetic alterations, highlighting these EACs as important environmental considerations for breast cancer research, clinical care and prevention

Homeostatic Imbalance and Colon Cancer: The Dynamic Epigenetic Interplay of Inflammation, Environmental Toxins, and Chemopreventive Plant Compounds

The advent of modern medicine has allowed for significant advances within the fields of emergency care, surgery, and infectious disease control. Health threats that were historically responsible for immeasurable tolls on human life are now all but eradicated within certain populations, specifically those that enjoy higher degrees of socio-economic status and access to healthcare. However, modernization and its resulting lifestyle trends have ushered in a new era of chronic illness; one in which an unprecedented number of people are estimated to contract cancer and other inflammatory diseases. Here, we explore the idea that homeostasis has been redefined within just a few generations, and that diseases such as colorectal cancer are the result of fluctuating physiological and molecular imbalances. Phytochemical-deprived, pro-inflammatory diets combined with low-dose exposures to environmental toxins, including bisphenol-A (BPA) and other endocrine disruptors, are now linked to increasing incidences of cancer in westernized societies and developing countries. There is recent evidence that disease determinants are likely set in utero and further perpetuated into adulthood dependent upon the innate and environmentally acquired phenotype unique to each individual. In order to address a disease as multi-factorial, case-specific, and remarkably adaptive as cancer, research must focus on its root causes in order to elucidate the molecular mechanisms by which they can be prevented or counteracted via plant-derived compounds such as epigallocatechin-3-gallate (EGCG) and resveratrol. The significant role of epigenetics in the regulation of these complex processes is emphasized here to form a comprehensive view of the dynamic interactions that influence modern-day carcinogenesis, and how sensibly restoring homeostatic balance may be the key to the cancer riddle

GSK-3 as potential target for therapeutic intervention in cancer

The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) was initially identified and studied in the regulation of glycogen synthesis. GSK-3 functions in a wide range of cellular processes. Aberrant activity of GSK-3 has been implicated in many human pathologies including: bipolar depression, Alzheimer's disease, Parkinson's disease, cancer, non-insulin-dependent diabetes mellitus (NIDDM) and others. In some cases, suppression of GSK-3 activity by phosphorylation by Akt and other kinases has been associated with cancer progression. In these cases, GSK-3 has tumor suppressor functions. In other cases, GSK-3 has been associated with tumor progression by stabilizing components of the beta-catenin complex. In these situations, GSK-3 has oncogenic properties. While many inhibitors to GSK-3 have been developed, their use remains controversial because of the ambiguous role of GSK-3 in cancer development. In this review, we will focus on the diverse roles that GSK-3 plays in various human cancers, in particular in solid tumors. Recently, GSK-3 has also been implicated in the generation of cancer stem cells in various cell types. We will also discuss how this pivotal kinase interacts with multiple signaling pathways such as: PI3K/PTEN/Akt/mTORC1, Ras/Raf/MEK/ERK, Wnt/beta-catenin, Hedgehog, Notch and others

Alteration of AKT Activity Increases Chemotherapeutic Drug and Hormonal Resistance in Breast Cancer yet Confers an Achilles Heel by Sensitization to Targeted Therapy

The PI3K/PTEN/Akt/mTOR pathway plays critical roles in the regulation of cell growth. The effects of this pathway on drug resistance and cellular senescence of breast cancer cells has been a focus of our laboratory. Introduction of activated Akt or mutant PTEN constructs which lack lipid phosphatase [PTEN(G129E)] or lipid and protein phosphatase [PTEN(C124S)] activity increased the resistance of the cells to the chemotherapeutic drug doxorubicin, and the hormonal drug tamoxifen. Activated Akt and PTEN genes also inhibited the induction of senescence after doxorubicin treatment; a phenomenon associated with unrestrained proliferation and tumorigenesis. Interference with the lipid phosphatase domain of PTEN was sufficient to activate Akt/mTOR/p70S6K as MCF-7 cells transfected with the mutant PTEN gene lacking the lipid phosphatase activity [PTEN(G129E)] displayed elevated levels of activated Akt and p70S6K compared to empty vector transfected cells. Cells transfected with mutant PTEN or Akt constructs were hypersensitive to mTOR inhibitors when compared with the parental or empty vector transfected cells. Akt-transfected cells were cultured for over two months in tamoxifen from which tamoxifen and doxorubicin resistant cells were isolated that were >10-fold more resistant to tamoxifen and doxorubicin than the original Akt-transfected cells. These cells had a decreased induction of both activated p53 and total p21Cip1 upon doxorubicin treatment. Furthermore, these cells had an increased inactivation of GSK-3β and decreased expression of the estrogen receptor-α. In these drug resistant cells, there was an increased activation of ERK which is associated with proliferation. These drug resistant cells were hypersensitive to mTOR inhibitors and also sensitive to MEK inhibitors, indicating that the enhanced p70S6K and ERK expression was relevant to their drug and hormonal resistance. Given that Akt is overexpressed in greater than 50% of breast cancers, our results point to potential therapeutic targets, mTOR and MEK. These studies indicate that activation of the Akt kinase or disruption of the normal activity of the PTEN phosphatase can have dramatic effects on activity of p70S6K and other downstream substrates and thereby altering the therapeutic sensitivity of breast cancer cells. The effects of doxorubicin and tamoxifen on induction of the Raf/MEK/ERK and PI3K/Akt survival pathways were examined in unmodified MCF-7 breast cells. Doxorubicin was a potent inducer of activated ERK and to a lesser extent Akt. Tamoxifen also induced ERK. Thus a consequence of doxorubicin and tamoxifen therapy of breast cancer is the induction of a pro-survival pathway which may contribute to the development of drug resistance. Unmodified MCF-7 cells were also sensitive to MEK and mTOR inhibitors which synergized with both tamoxifen and doxorubicin to induce death. In summary, our results point to the key interactions between the PI3K/PTEN/Akt/mTOR and Raf/ MEK/ERK pathways in regulating chemotherapeutic drug resistance/sensitivity in breast cancer and indicate that targeting these pathways may prevent drug and hormonal resistance. Orignally published Advances in Enzyme Regulation, Vol. 48, No. 1, 2008

The Role of GSK-3[beta] in MCF-7 Breast Cancer Cell Signaling and Drug Resistance

Glycogen synthase kinase- 3beta (GSK-3beta) is well documented to participate in a complex array of critical cellular processes. This versatile protein is involved in numerous signaling pathways that influence metabolism, embryogenesis, differentiation, migration, cell cycle progression, and survival. Aberrant activity of GSK-3beta has been implicated in pathologies such as type-2 diabetes, bipolar disorder, Alzheimer's, and cancer. GSK-3beta is normally active in the cytoplasm of resting cells in an unphosphorylated state where it suppresses an assortment of transcription factors implicated in oncogenesis. Lying downstream of the PI3K/PTEN/Akt pathway, GSK-3beta can be negatively regulated through phosphorylation by active Akt. Given this pathway's role in malignant transformation, prevention of apoptosis, drug resistance, and metastasis, this study was performed to elucidate the role of GSK-3beta in MCF-7 breast cancer cells. It was found that cells expressing a kinase dead (KD) form of GSK-3beta were more resistant to doxorubicin and tamoxifen, as well as highly clonogenic compared to cells harboring wild-type (WT) or constitutively active (A9) GSK-3beta. However, when treated with rapamycin, GSK-3beta KD cells show a marked decrease in proliferation as compared to WT or A9 cells. Additionally, resistance to doxorubicin and tamoxifen were alleviated in KD cells upon co-treatment with the Array MEK inhibitor. Taken together, these results suggest that the loss of GSK-3beta activity in MCF-7 breast cancer cells promotes clonogenicity and drug resistance, but sensitizes the cells to signaling pathway blockade. Therefore, targeting aberrant activity of the PI3K/PTEN/Akt/GSK-3beta pathway may be a clinically relevant tool for both increasing efficacy of and avoiding resistance to conventional therapy.  M.S

The Role of GSK-3[beta] in MCF-7 Breast Cancer Cell Signaling and Drug Resistance

Glycogen synthase kinase- 3beta (GSK-3beta) is well documented to participate in a complex array of critical cellular processes. This versatile protein is involved in numerous signaling pathways that influence metabolism embryogenesis differentiation migration cell cycle progression and survival. Aberrant activity of GSK-3beta has been implicated in pathologies such as type-2 diabetes bipolar disorder Alzheimer's and cancer. GSK-3beta is normally active in the cytoplasm of resting cells in an unphosphorylated state where it suppresses an assortment of transcription factors implicated in oncogenesis. Lying downstream of the PI3K/PTEN/Akt pathway GSK-3beta can be negatively regulated through phosphorylation by active Akt. Given this pathway's role in malignant transformation prevention of apoptosis drug resistance and metastasis this study was performed to elucidate the role of GSK-3beta in MCF-7 breast cancer cells. It was found that cells expressing a kinase dead (KD) form of GSK-3beta were more resistant to doxorubicin and tamoxifen as well as highly clonogenic compared to cells harboring wild-type (WT) or constitutively active (A9) GSK-3beta. However when treated with rapamycin GSK-3beta KD cells show a marked decrease in proliferation as compared to WT or A9 cells. Additionally resistance to doxorubicin and tamoxifen were alleviated in KD cells upon co-treatment with the Array MEK inhibitor. Taken together these results suggest that the loss of GSK-3beta activity in MCF-7 breast cancer cells promotes clonogenicity and drug resistance but sensitizes the cells to signaling pathway blockade. Therefore targeting aberrant activity of the PI3K/PTEN/Akt/GSK-3beta pathway may be a clinically relevant tool for both increasing efficacy of and avoiding resistance to conventional therapy.

Involvement of Akt-1 and mTOR in sensitivity of breast cancer to targeted therapy

Elucidating the response of breast cancer cells to chemotherapeutic and hormonal based drugs is clearly important as these are frequently used therapeutic approaches. A signaling pathway often involved in chemo- and hormonal-resistance is the Ras/PI3K/PTEN/Akt/mTOR cascades. In the studies presented in this report, we have examined the effects of constitutive activation of Akt on the sensitivity of MCF-7 breast cancer cells to chemotherapeutic- and hormonal-based drugs as well as mTOR inhibitors. MCF-7 cells which expressed a constitutively-activated Akt-1 gene [∆Akt-1(CA)] were more resistant to doxorubicin, etoposide and 4-OH-tamoxifen (4HT) than cells lacking ∆Akt-1(CA). Cells which expressed ∆Akt-1(CA) were hypersensitive to the mTOR inhibitor rapamycin. Furthermore, rapamycin lowered the IC50s for doxorubicin, etoposide and 4HT in the cells which expressed ∆Akt-1(CA), demonstrating a potential improved method for treating certain breast cancers which have deregulated PI3K/PTEN/Akt/mTOR signaling. Understanding how breast cancers respond to chemo- and hormonal-based therapies and the mechanisms by which they can become drug resistant may enhance our ability to treat breast cancer. These results also document the potential importance of knowledge of the mutations present in certain cancers which may permit more effective therapies

Fusogenic-Oligoarginine Peptide-Mediated Delivery of siRNAs Targeting the CIP2A Oncogene into Oral Cancer Cells

<div><p>Despite a better understanding of the pathogenesis of oral cancer, its treatment outcome remains poor. Thus, there is a need for new therapeutic strategies to improve the prognosis of this disease. RNA interference (RNAi) appears to be a promising therapeutic tool for the treatment of many diseases, including oral cancer. However, an obstacle for RNAi-mediated therapies has been delivery, in particular, the retention of small interfering RNAs (siRNAs) in endosomes and their subsequent degradation in lysosomes, resulting in inefficient gene silencing. Thus, the current study examined the feasibility of designing and utilizing a peptide, termed 599, consisting of a synthetic influenza virus-derived endosome-disruptive fusogenic peptide sequence and a stretch of cationic cell-penetrating nona(D-arginine) residues, to deliver siRNAs into oral cancer cells and induce silencing of the therapeutic target, CIP2A, an oncoprotein overexpressed in various human malignancies including oral cancer. Increasing the 599 peptide-to-siRNA molar ratio demonstrated a higher binding capacity for siRNA molecules and enhanced siRNA delivery into the cytoplasm of oral cancer cells. In fact, quantitative measurements of siRNA delivery into cells demonstrated that a 50∶1 peptide-to-siRNA molar ratio could deliver 18-fold higher amounts of siRNAs compared to cells treated with siRNA alone with no significant long-term cytotoxic effects. Most importantly, the 599 peptide-mediated siRNA delivery promoted significant CIP2A mRNA and protein silencing which resulted in decreased oral cancer cell invasiveness and anchorage-independent growth. Together, these data demonstrate that a chimeric peptide consisting of a fusogenic sequence, in combination with cell-penetrating residues, can be used to effectively deliver siRNAs into oral cancer cells and induce the silencing of its target gene, potentially offering a new therapeutic strategy in combating oral cancer.</p></div

Optimization of 599 peptide binding to siRNAs.

<p>An ethidium bromide stained 4% agarose gel shift assay examining the ability of various amounts of the 599 peptide (ranging from 1 to 50-fold molar excess of siRNAs) to form complexes with siCIP2A. siCIP2A, siRNA targeting the CIP2A oncogene; MWM, molecular weight marker (the number of base pairs for each DNA fragment are shown).</p

Particle characterization of the 599/siCIP2A complex.

<p>(<b>A</b>) Size distribution and zeta potential of the 599 peptide complexed with siCIP2A at a 50∶1 peptide-to-siRNA molar ratio 20 minutes after formulation in water. (<b>B</b>) Darkfield-based optical microscopy image of the 599 peptide complexed with siCIP2A at a 50∶1 peptide-to-siRNA molar ratio 20 minutes after formulation in water. Scale bar: 10,000 nm.</p