Correction: Phaeosphaeride A Isolated from an Endophytic Paraphoma sp. Alleviates ABCG2-mediated Resistance to Mitoxantrone in Breast Cancer Cells.

Both 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F-203; NSC 703786) and 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (GW-610; NSC 721648) are antitumor agents with novel mechanism(s). Previous studies have indicated that cytochrome (CYP) P450 1A1 is crucial for 5F-203 activity. In the present study, we investigated the functional role of 2 newly identified CYP P450 enzymes, CYP2S1 and CYP2W1, in mediating antitumor activity of benzothiazole compounds. We generated isogenic breast cancer (MDA-MB-468, MCF-7) and colorectal cancer (CRC; KM12 and HCC2998) cell lines depleted for CYP1A1, CYP2S1, or CYP2W1. The sensitivity of these cells to 5F-203 and GW-610 was then compared with vector control cells. 5F-203 exhibited potent activity against breast cancer cells, whereas GW-610 was effective against both breast and colorectal cancer cells. CYP1A1 was induced in both breast cancer and CRC cells, while CYP2S1 and CYP2W1 were selectively induced in breast cancer cells only following treatment with 5F-203 or GW-610. Depletion of CYP1A1 abrogated the sensitivity of breast cancer and CRC cells to 5F-203 and GW-610. Although depletion of CYP2S1 sensitized both breast cancer and CRC cells toward 5F-203 and GW-610, CYP2W1 knockdown caused marked resistance to GW-610 in CRC cells. Our results indicate that CYP-P450 isoforms, with the exception of CYP1A1, play an important role in mediating benzothiazole activity. CYP2S1 appears to be involved in deactivation of benzothiazoles, whereas CYP2W1 is important for bioactivation of GW-610 in CRC cells. Because CYP2W1 is highly expressed in colorectal tumors, GW-610 represents a promising agent for CRC therapy.

<div>Abstract<p>Both 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F-203; NSC 703786) and 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (GW-610; NSC 721648) are antitumor agents with novel mechanism(s). Previous studies have indicated that cytochrome (CYP) P450 1A1 is crucial for 5F-203 activity. In the present study, we investigated the functional role of 2 newly identified CYP P450 enzymes, CYP2S1 and CYP2W1, in mediating antitumor activity of benzothiazole compounds. We generated isogenic breast cancer (MDA-MB-468, MCF-7) and colorectal cancer (CRC; KM12 and HCC2998) cell lines depleted for CYP1A1, CYP2S1, or CYP2W1. The sensitivity of these cells to 5F-203 and GW-610 was then compared with vector control cells. 5F-203 exhibited potent activity against breast cancer cells, whereas GW-610 was effective against both breast and colorectal cancer cells. CYP1A1 was induced in both breast cancer and CRC cells, while CYP2S1 and CYP2W1 were selectively induced in breast cancer cells only following treatment with 5F-203 or GW-610. Depletion of CYP1A1 abrogated the sensitivity of breast cancer and CRC cells to 5F-203 and GW-610. Although depletion of CYP2S1 sensitized both breast cancer and CRC cells toward 5F-203 and GW-610, CYP2W1 knockdown caused marked resistance to GW-610 in CRC cells. Our results indicate that CYP-P450 isoforms, with the exception of CYP1A1, play an important role in mediating benzothiazole activity. CYP2S1 appears to be involved in deactivation of benzothiazoles, whereas CYP2W1 is important for bioactivation of GW-610 in CRC cells. Because CYP2W1 is highly expressed in colorectal tumors, GW-610 represents a promising agent for CRC therapy. <i>Mol Cancer Ther; 10(10); 1982–92. ©2011 AACR</i>.</p></div>

<div>Abstract<p>Both 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F-203; NSC 703786) and 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (GW-610; NSC 721648) are antitumor agents with novel mechanism(s). Previous studies have indicated that cytochrome (CYP) P450 1A1 is crucial for 5F-203 activity. In the present study, we investigated the functional role of 2 newly identified CYP P450 enzymes, CYP2S1 and CYP2W1, in mediating antitumor activity of benzothiazole compounds. We generated isogenic breast cancer (MDA-MB-468, MCF-7) and colorectal cancer (CRC; KM12 and HCC2998) cell lines depleted for CYP1A1, CYP2S1, or CYP2W1. The sensitivity of these cells to 5F-203 and GW-610 was then compared with vector control cells. 5F-203 exhibited potent activity against breast cancer cells, whereas GW-610 was effective against both breast and colorectal cancer cells. CYP1A1 was induced in both breast cancer and CRC cells, while CYP2S1 and CYP2W1 were selectively induced in breast cancer cells only following treatment with 5F-203 or GW-610. Depletion of CYP1A1 abrogated the sensitivity of breast cancer and CRC cells to 5F-203 and GW-610. Although depletion of CYP2S1 sensitized both breast cancer and CRC cells toward 5F-203 and GW-610, CYP2W1 knockdown caused marked resistance to GW-610 in CRC cells. Our results indicate that CYP-P450 isoforms, with the exception of CYP1A1, play an important role in mediating benzothiazole activity. CYP2S1 appears to be involved in deactivation of benzothiazoles, whereas CYP2W1 is important for bioactivation of GW-610 in CRC cells. Because CYP2W1 is highly expressed in colorectal tumors, GW-610 represents a promising agent for CRC therapy. <i>Mol Cancer Ther; 10(10); 1982–92. ©2011 AACR</i>.</p></div>

Spontaneous Formation of Tumorigenic Hybrids between Breast Cancer and Multipotent Stromal Cells Is a Source of Tumor Heterogeneity

We are seeking to enhance radiation and chemotherapies that are currently being used to treat lung and breast cancers to improve the survival of people afflicted with these deadly diseases. For sensitizing lung and breast cancer cells we use two agents that both increase metabolic oxidative stress by disrupting mitochondrial function. One is a positively charged molecule with a long hydrophobic chain that inserts into the mitochondrial membrane, decreasing the mitochondrial membrane potential and increasing oxidation. The other agent inhibits mitochondrial pyruvate import and also results in increased metabolic oxidative stress.We hypothesized that disrupting the mitochondrial membrane potential of cancer cells would result in increasing steady-state levels of mitochondrial reactive oxygen species (ROS) to induce a toxic level of oxidative stress in breast cancer cells, relative to non-malignant cells, when combined with inhibition of hydroperoxide metabolism. Treatment with Decyl-Triphenylphosphonium (DTPP, 1 μM), a lipophilic cation that localizes to cancer cell mitochondria, caused an increase in generalized oxidation (CDCFH2) as well as an increase in oxidation localized to the mitochondria (MitoSOX) that was specific to cancer cells. In addition, DTPP resulted in an increase in oxidized glutathione and thioredoxin reductase activity. DTPP induced oxidation and cytotoxicity was enhanced by glutathione depletion (using buthionine sulfoximine, BSO 100 μM) as well as inhibition of thioredoxin reductase (using Auranofin, AUR 500 nM) in breast cancer cells. N-acetylcysteine (NAC), a non-specific thiol antioxidant, and PEG-SOD+PEG-CAT could protect against toxicity of BSO, AUR and DTPP exposure confirming a thiol-mediated oxidative stress mechanism of cell death. In another approach we targeted the import of pyruvate into the mitochondria with a small molecule inhibitor of the mitochondrial pyruvate carrier (MPC), UK5099. Treatment with 5 μM UK5099 to inhibit the MPC selectively sensitized lung and breast cancer cells to clonogenic cell killing when combined with depletion of glutathione, relative to non-malignant lung and breast epithelial cells. Furthermore, cancer cell killing mediated by UK5099 combined with BSO was inhibited by the thiol antioxidant NAC, independent of glutathione levels indicating a mechanism of toxicity involving thiol redox state and metabolic oxidative stress. Using oxidation sensitive fluorescent dyes (CDCFH2 and MitoSOX), treatment with UK5099 induced increases in steady state levels of pro-oxidants which were further increased with BSO treatment. The proposed mechanism is mitochondrial hydroperoxides and superoxide that were inhibited by pegylated-SOD and induced overexpression of mitochondrial targeted catalase but not cytosolic catalase. Treatment of breast and non-small cell lung cancer cells with UK5099 significantly sensitized cancer cells to clonogenic cell killing mediated by paclitaxel, carboplatin, or etoposide. Treatment with UK5099+BSO for 48 hours significantly sensitized breast and non-small cell lung cancer cells, but not non-malignant breast or lung epithelial cells, to 6 Gy radiation in a single dose, with NAC ameliorating the toxicity of UK5099+BSO+radiation. The use of doxycycline inducible expression of antioxidant enzyme for mitochondrial targeted catalase in breast and lung cancer cell lines has shown mechanistic protection of UK5099 toxicity caused by H2O2 at the mitochondria of cancer cells. Finally, CRISPR knockout of MPC1 recapitulates the UK5099-induced sensitization to both BSO or BSO+radiation treatment and is ameliorated by NAC. These data support the hypothesis that disrupting the mitochondrial membrane potential with either a charged molecule targeted to electron transport chain (ETC) activity or MPC inhibitor selectively increases metabolic oxidation in cancer versus normal cells. Furthermore, these results also support the hypothesis that inhibition of the MPC could represent a significant target for sensitizing human breast and lung cancer cells to chemotherapy and radiation induced oxidative stress.

Breast cancer preferentially metastasizes to the bone, where the five-year relative survival rate falls from 90% to <10%. Although the precise mechanism underlying preferential metastasis is unknown, the bone likely provides a hospitable environment that both attracts breast cancer cells and allows them to colonize and grow. Besides affects osteoblast and osteoclast properties, we have evidence that metastatic breast cancer cells further create a unique bone niche by orchestrating extensive crosstalk with osteoblasts that may be involved in creating a niche permissive for breast cancer cell dormancy. MC3T3-E1 murine osteoblasts were grown to confluence, then MDA-MB-231 human breast cancer cells added at a ratio of 1 breast cancer cell to 10 osteoblasts. Co-cultures were subsequently fixed and stained for Cx43 expression. Next, MC3T3-E1 cells and MDA-MB-231 cells were grown to confluence. Culture supernatants were removed and processed through a series of ultra-centrifugation, purification, and concentration steps to purify for exosomes. Finally, MC3T3-E1 cells were grown to various stages of maturity: 4 (growth) and 10 days (early differentiation) and incubated with conditioned medium from human MDA-MB-231 cells. qPCR was used to assay for changes in osteoblast-derived inflammatory cytokine expression. Expression of the gap junction protein Cx43 was localized between breast cancer cells and osteoblasts. Both MC3T3-E1 and MDA-MB-231 cells expressed exosomes, which were visualized using a transmission electron microscope. Osteoblast-derived cytokines increased in the presence of MDA-MB-231 breast cancer cell conditioned medium included MCP-1 (CCL2), LIX (CXCL5), LIF, and NFATc2. Overall, these data suggest that suggest there is extensive crosstalk directly between osteoblasts and breast cancer cells, and that osteoblasts are an important source of cytokines in breast cancer bone metastasis. Thus, these findings implicate the bone microenvironment and cancer cell manipulation thereof in orchestrating metastatic tumor cell dormancy, colonization, and survival. Supported in part by the National Institutes of Health; 1RC1 CA146381, 1R01NS06994, P50 CA083639 for FCM; Ruth L. Kirschstein Institutional Research Service Award (NRSA) T32 CA079448 for KMB. Citation Format: Karen M. Bussard, Frank C. Marini. Crosstalk between breast cancer and stromal cells occurs via exosomes and gap junctions in bone metastatic breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1625. doi:10.1158/1538-7445.AM2013-1625

Aberrant activity of the phosphatidylinositol 3-kinase (PI3K) pathway supports growth of many tumors including those of breast, lung, and prostate. Resistance of breast cancer cells to targeted chemotherapies including tyrosine kinase inhibitors (TKI) has been linked to persistent PI3K activity, which may in part be due to increased membrane expression of epidermal growth factor (EGF) receptors (HER2 and HER3). Recently we found that proteins of the RGS (regulator of G protein signaling) family suppress PI3K activity downstream of the receptor by sequestering its p85alpha subunit from signaling complexes. Because a substantial percentage of breast tumors have RGS16 mutations and reduced RGS16 protein expression, we investigated the link between regulation of PI3K activity by RGS16 and breast cancer cell growth. RGS16 overexpression in MCF7 breast cancer cells inhibited EGF-induced proliferation and Akt phosphorylation, whereas shRNA-mediated extinction of RGS16 augmented cell growth and resistance to TKI treatment. Exposure to TKI also reduced RGS16 expression in MCF7 and BT474 cell lines. RGS16 bound the amino-terminal SH2 and inter-SH2 domains of p85alpha and inhibited its interaction with the EGF receptor-associated adapter protein Gab1. These results suggest that the loss of RGS16 in some breast tumors enhances PI3K signaling elicited by growth factors and thereby promotes proliferation and TKI evasion downstream of HER activation.

Bone is the most common site to which breast cancer cells metastasize. We found that osteoblast-like MG63 cells and human bone tissue contain the bile acid salt sodium deoxycholate (DC). MG63 cells take up and accumulate DC from the medium, suggesting that the bone-derived DC originates from serum. DC released from MG63 cells or bone tissue promotes cell survival and induces the migration of metastatic human breast cancer MDA-MB-231 cells. The bile acid receptor farnesoid X receptor (FXR) antagonist Z-guggulsterone prevents the migration of these cells and induces apoptosis. DC increases the gene expression of FXR and induces its translocation to the nucleus of MDA-MB-231 cells. Nuclear translocation of FXR is concurrent with the increase of urokinase-type plasminogen activator (uPA) and the formation of F-actin, two factors critical for the migration of breast cancer cells. Our results suggest a novel mechanism by which DC-induced increase of uPA and binding to the uPA receptor of the same breast cancer cell self-propel its migration and metastasis to the bone.

Adenoviral vectors with E1A regulated by tumor-specific promoters are selectively cytolytic for breast cancer and melanoma.

Impact of cancer stem cell markers Nestin and Sox2 on cell migration of epithelial and mesenchymal pancreatic cancer cells

Simple SummaryBreast cancer accounts as the most extended disease among women worldwide. Store-operated calcium entry (SOCE), a major mechanism that allows calcium entry from the extracellular region through the plasma membrane, is required for several physiological processes. In recent years, it has been revealed that several breast cancer types present dysregulated calcium homeostasis, which contribute to their malignancy. Here we show the role of two important regulators of SOCE, SARAF and EFHB, which are necessary for cell viability, proliferation, and migration in breast cancer and pre-neoplastic cells, respectively, thus suggesting that these regulators play a key function in breast cancer development and progression.Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Store-operated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in luminal and triple negative breast cancer (TNBC) cells and is essential for full SOCE in these cells. SARAF expression was found to be similar in breast cancer and pre-neoplastic breast epithelial cells, and SARAF knockdown was found to result in enhanced SOCE in pre-neoplastic and TNBC cells. Interestingly, silencing SARAF expression in ER+ MCF7 cells led to attenuation of SOCE, thus suggesting a distinctive role for SARAF in this cell type. Finally, we used a combination of approaches to show that molecular knockdown of SARAF and EFHB significantly attenuates the ability of breast cancer cells to proliferate and migrate, as well as cell viability. In aggregate, SARAF and EFHB are required for the fine modulation of SOCE in breast cancer cells and play an important role in the maintenance of proliferation, migration, and viability in these cells.

World Health Organization articulated 9.8 million casualties globally in 2018 due to cancer. Cancer, as the world's second most fatal disease, can be recuperated well if diagnosed at an early stage. In this work, a gradient-based impedance synthesis of normal and cancerous cells of breast and lungs, is demonstrated numerically for early-stage cancer detection. Low-voltage single-cell level examination is employed for indomitable diagnosis. MCF-7 and MCF-10A are utilized as breast cancer and breastnormal cells, respectively; likewise, SK-MES and NL-20 are utilized as lung cancer and lung normal cell. Pre-examination numerical setup validity ensured with multiple test regimes. Micro-scaled planar and nano-structured electrodes are employed individually to witness the effect of the electrode's structure during electrical impedance examination of cancer and non-cancer cell. Frequency range, at which differential impedance effect is found detectable, for breast and lung cancer cell pairs is determined to be 107Hz and 108Hz, respectively. By surpassing the conventional impedance spectroscopy with tedious data fitting formalities, the gradient synthesis technique for cancer detection is introduced. The gradient synthesis for cancer detection is found independent of electrode shape effect. Gradient for breast cancer cell is found to be 2 times greater than the normal breast cell while for lung cancer cell it is found to be 1.5 times greater than the normal lung cell. Our results suggest that as the frequency of applied electrical stimulus increases, impedance of cancerous cell falls at the rate almost double than its counterpart normal cell. This work provides a theoretical basis for further experimental exploration of gradient-based impedance synthesis in cancer therapy and serves as a design tool for performance optimization. Figure 1 (a) Represents electrical Impedance analysis of breast normal cell MCF-10A and breast cancer cell MCF-7 using micro-scaled planar and nano-structured electrodes. (b) Gradient impedance synthesis performed, for breast normal cell (MCF-10A) and breast cancer cell (MCF-7) likewise for lungs normal cell (NL-20) and lungs cancer cell (SK-MES), which assures clear differential effect for cancer screening. Surpassing the conventional and tedious data fitting impedance spectroscopies, a novel gradient-based impedance spectroscopy for early cancer detection is introduced. It clearly detects cancer without any data fitting formalities to find parameter of identification. Planar and nano structure electrodes are used to witness the impact of electrode shape on cell impedance. Breast normal MCF-10A and cancer cell MCF-7 as well as lungs normal NL-20 and cancer cell SK-MES are examined to reflect the efficacy of our work. Single cell level examination is performed for authenticated results.

BackgroundZinc transport proteins (ZIP and ZnT), metallothioneins (MT) and protein kinase CK2 are involved in dysregulation of zinc homeostasis in breast and prostate cancer cells. Following up our previous research, we targeted ZIP12, ZnT1, MT2A and CK2 in this study by investigating their expression levels and protein localisation. MethodsQuantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence confocal microscopy were employed to quantify the expression of ZIP12, ZnT1, MT2A and CK2 subunits in a panel of breast and prostate cell lines without or with extracellular zinc exposure. The cellular localisations of these target proteins were also examined by immunofluorescence confocal microscopy. ResultsIn response to the extracellular zinc exposure, the gene expression was elevated for SLC39A12 (ZIP12), SLC30A1 (ZnT1) and MT2A (MT2A) in normal prostate epithelial cells (RWPE-1) in contrast to their cancerous counterparts (PC3 and DU145), whilst the gene expression was higher for SLC39A12 (ZIP12) and SLC30A1 (ZnT1) in both normal (MCF10A) and basal breast cancer cells (MDA-MB-231) compared to luminal breast cancer cells (MCF-7). At the protein level, the expression for both ZIP12 and ZnT1 was trending lower in the time course for the breast cancer cells whilst their expression was remained constant in the normal breast epithelial cells. The expression of ZIP12 in prostate cancer cells was higher than the normal prostate cells. The protein expression for CK2 α/αꞌ and CK2β was markedly higher in prostate cancer cells than the normal prostate cells. Upon extracellular zinc exposure, ZIP12 was, for the first time, conspicuously localised in the plasma membrane of breast cancer cells but not in normal breast epithelial cells and prostate cells. ZnT1 is only localised in the plasma membrane of breast cancer cells. MT2A is distinctively seen close to the plasma membrane in breast cancer cells. CK2 is also for the first time shown to be localised in proximity to the plasma membrane of breast cancer cells. ConclusionThe findings, particularly the localisation of ZIP12 and CK2, are novel and significant for our understanding of zinc homeostasis in breast and prostate cancer cells.

Autophagy is an alternative cell death pathway that is induced by mammalian target of rapamycin (mTOR) inhibitors and up-regulated when apoptosis is defective. We investigated radiation-induced autophagy in the presence or absence of Bax/Bak with or without an mTOR inhibitor, Rad001. Two isogenic cell lines, wild type (WT) and Bak/Bak(-/-) mouse embryonic fibroblasts and tumor cell lines were used for this study. Irradiated Bak/Bak(-/-) cells had a decrease of Akt/mTOR signaling and a significant increase of pro-autophagic proteins ATG5-ATG12 COMPLEX and Beclin-1. These molecular events resulted in an up-regulation of autophagy. Bax/Bak(-/-) cells were defective in undergoing apoptosis but were more radiosensitive than the WT cells in autophagy. Both autophagy and sensitization of Bak/Bax(-/-) cells were further enhanced in the presence of Rad001. In contrast, inhibitors of autophagy rendered the Bak/Bax(-/-) cells radioresistant, whereas overexpression of ATG5 and Beclin-1 made the WT cells radiosensitive. When this novel concept of radiosensitization was tested in cancer models, small interfering RNAs against Bak/Bax also led to increased autophagy and sensitization of human breast and lung cancer cells to gamma radiation, which was further enhanced by Rad001. This is the first report to demonstrate that inhibition of pro-apoptotic proteins and induction of autophagy sensitizes cancer cells to therapy. Therapeutically targeting this novel pathway may yield significant benefits for cancer patients.

Integrins contribute to progression in many cancers, including breast cancer. For example, the interaction of alpha(5)beta(1) with plasma fibronectin causes the constitutive invasiveness of human prostate cancer cells. Inhibition of this process reduces tumorigenesis and prevents metastasis and recurrence. In this study, naturally serum-free basement membranes were used as invasion substrates. Immunoassays were used to compare the roles of alpha(5)beta(1) and alpha(4)beta(1) fibronectin receptors in regulating matrix metalloproteinase (MMP)-1-dependent invasion by human breast cancer and mammary epithelial cells. We found that a peptide consisting of fibronectin PHSRN sequence, Ac-PHSRN-NH(2), induces alpha(5)beta(1)-mediated invasion of basement membranes in vitro by human breast cancer and mammary epithelial cells. PHSRN-induced invasion requires interstitial collagenase MMP-1 activity and is suppressed by an equimolar concentration of a peptide consisting of the LDV sequence of the fibronectin connecting segment, Ac-LHGPEILDVPST-NH(2), in mammary epithelial cells, but not in breast cancer cells. This sequence interacts with alpha(4)beta(1), an integrin that is often down-regulated in breast cancer cells. Immunoblotting shows that the PHSRN peptide stimulates MMP-1 production by serum-free human breast cancer and mammary epithelial cells and that the LDV peptide represses PHSRN-stimulated MMP-1 production only in mammary epithelial cells. Furthermore, PHSRN stimulates MMP-1 activity in breast cancer cells and mammary epithelial cells with a time course that closely parallels invasion induction. Thus, down-regulation of surface alpha(4)beta(1) during oncogenic transformation may be crucial for establishment of the alpha(5)beta(1)-induced, MMP-1-dependent invasive phenotype of breast cancer cells.