Nature’s touch in oncology: evidence based approach exploring phytoscience for emerging therapeutics of breast cancer

Breast cancer development and progression: Risk factors, cancer stem cells, signaling pathways, genomics, and molecular pathogenesis

Parity (childbearing) significantly decreases a woman’s risk of breast cancer (BCa). Two proposed mechanisms of parity-induced protection investigated herein are a reduction in mammary stem cells (MaSCs) or an increase in differentiation in the parous mammary gland. MaSCs are postulated to mediate BCa risk as they are long-lived allowing a greater chance of accumulating genetic mutations likely to facilitate a carcinogenic transformation. Thus, a reduction in MaSCs by pregnancy may equate to a reduced risk of BCa. In order to investigate this, I first set out to ensure the isolation method used was highly selective for MaSCs. Cells expressing the proposed stem cell marker Stem Cell Antigen 1 (Sca-1) were characterized, as preliminary data indicated its superior ability to enrich for MaSCs beyond that of classical markers (defined by heat-stable antigen (CD24) positivity and high expression of integrin alpha 6 (CD49f)). Then, analyses of DNA damage markers and in vitro repair responses were used to assess the carcinogen sensitivity of MaSCs following treatment with a chemical carcinogen. Finally, primary and serial mammary fat pad stem cell assays were used to assess MaSC numbers and activity following pregnancy. Sca-1 was found not to further enrich for MaSC activity with a higher selectivity than the classical markers of MaSCs, thus CD24+CD49fhi cells were assessed for carcinogen sensitivity and stem cell activity following pregnancy. CD24+CD49fhi MaSC-enriched cells were found to be susceptible to carcinogens but not reduced by parity. Rather, parity appears to reduce a rare subset of cells that display stem cell activity outside the classical CD24+CD49fhi MaSC-enriched population. The terminal differentiation of the mammary gland that occurs at pregnancy is believed to permanently alter the gene expression profile. Preliminary data indicated the cell-specific target of these gene expression changes by parity was the stromal fibroblasts. The gene expression changes suggested a reduction in proliferative ability. To confirm this, the in vitro proliferative rate of parous fibroblasts was assessed in comparison to age-matched nulliparous fibroblasts. To then see if this proliferative rate was capable of affecting the proliferation of adjacent BCa cells, fibroblasts were co-cultured with low and high grade BCa cells in 3D matrices. Finally the ability of the parous fibroblasts to regulate in vivo tumour formation was assessed by co-injecting fibroblasts and BCa cells into mice. Parity was observed to reduce the proliferative rate in primary cultures of fibroblasts in 50% of the batches isolated over the course of the study. When tested using in vitro 3D proliferation assays or in vivo tumour growth assays, the effect of the parous fibroblasts was equally variable. In contrast, nulliparous fibroblasts were shown to be capable of tumour-stimulating behaviour on several occasions, an effect not observed in their parous counterparts. Altogether this work shows that a non-classical stem cell within the CD24+CD49lo/med population is decreased with parity and parous mammary fibroblasts have a less tumour stimulatory phenotype. This work thus identifies two cell types to further explore with the long-term goal to develop preventative therapies mimicking parity protection against BCa.

Environmental noise and breast cancer risk?

There is compelling evidence from transgenic mouse studies and analysis of mutations in human carcinomas indicating that the TGF-β signal transduction pathway is tumor suppressive.We have shown that overexpression of TGF-β1 in mammary epithelial cells suppresses the development of carcinomas and that expression of a dominant negative type II TGF-β receptor (DNIIR) in mammary epithelial cells under control of the MMTV promoter/enhancer increases the incidence of mammary carcinomas.Studies of human tumors have demonstrated inactivating mutations in human tumors of genes encoding proteins involved in TGF-β signal transduction, including DPC4/Smad4, Smad2, and the type II TGF-β receptor (TβRII).There is also evidence that TGF-β can enhance the progression of tumors.This hypothesis is being tested in genetically modified mice.To attain complete loss of TβRII, we have generated mice with loxP sites flanking exon 2 of Tgfbr2 and crossed them with mice expressing Cre recombinase under control of the MMTV promoter/enhancer to obtain Tgfbr2 mgKO mice.These mice show lobuloalveolar hyperplasia.Mice are being followed for mammary tumor development.Tgfbr2 mgKO mice that also express polyoma virus middle T antigen under control of the MMTV promoter (MMTV-PyVmT) develop mammary tumors with a significantly shorter latency than MMTV-PyVmT mice and show a marked increase in pulmonary metastases.Our data do not support the hypothesis that TGF-β signaling in mammary carcinoma cells is important for invasion and metastasis, at least in this model system.The importance of stromal-epithelial interactions in mammary gland development and tumorigenesis is well established.These interactions probably involve autocrine and paracrine action of multiple growth factors, including members of the TGF-β family, which are expressed in both stroma and epithelium.Again, to accomplish complete knockout of the type II TGF-β receptor gene in mammary stromal cells, FSP1-Cre and Tgfbr2 flox/flox mice were crossed to attain Tgfbr2 fspKO mice.The loss of TGF-β responsiveness in fibroblasts resulted in intraepithelial neoplasia in prostate and invasive squamous cell carcinoma of the forestomach with high penetrance by 6 weeks of age.Both epithelial lesions were associated with an increased abundance of stromal cells.Activation of paracrine hepatocyte growth factor (HGF) signaling was identified as one possible mechanism for stimulation of epithelial proliferation.TGF-β signaling in fibroblasts thus modulates the growth and oncogenic potential of adjacent epithelia in selected tissues.More recently, we have examined the effects of Tgfbr2 fspKO fibroblasts on normal and transformed mammary epithelium.We analyzed the role of TGF-β signaling by stromal cells in mammary tumor progression.To avoid the possibility of endogenous wild-type fibroblasts masking potential effects of Tgfbr2 fspKO cells on tumor progression, we implanted PyVmT mammary carcinoma cells with Tgfbr2 fspKO or wildtype fibroblasts in the subrenal capsule of nude mice.Mammary tumor cells implanted with Tgfbr2 fspKO cells exhibited an increase in tumor growth and intravasation associated with an increase in tumor cell survival, proliferation and an increase in tumor angiogenesis compared with tumor cells implanted with control fibroblasts.We demonstrated increased expression of several growth factors by Tgfbr2 fspKO fibroblasts compared with control fibroblasts in primary culture.These included HGF, MSP and TGF-α.There was an increase in tumor cell activating phosphorylation of the cognate receptors, c-Met, RON, erbB1, and erbB2 in carcinomas accompanied by Tgfbr2 fspKO fibroblasts.The Tgfbr2 fspKO mouse model illustrates that a signaling pathway known to suppress cell-cycle progression when activated in epithelial cells can also have an indirect inhibitory effect on epithelial proliferation when activated in adjacent stromal fibroblasts in vivo.Loss of this inhibitory effect can result in increased epithelial proliferation and may even progress to invasive carcinoma in some tissues.

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.

Breast cancer occurs at rate of 1 out of 7. After so many years of research, even though treatment has been dramatically improved and survival rate and length significantly increased, there is still not cure for the disease. The possible existence of mammary gland stem cell and breast cancer stem cell 1, 2 lights the hope for finding a cure to this deadly disease. The rational behind is that once the stem cell can be isolated, the signalling pathways that regulate its proliferation, self-renewal, survival and differentiation will be illustrated, and this might shed light on the mechanisms of breast cancer formation, therefore leading to a better therapeutic treatment. Existence of stem cell in normal mammary gland has been convincingly demonstrated by Kordon and Smith 1. In their report, an entire mammary gland could be regenerated with the progeny of a single cell following transplantation into cleared mammary fat pads. By definition, mammary gland stem cells are those that rarely divide and persist throughout reproductive life. Classical markers 3 for identifying and purifying mammary stem cells are label retention (tritiated thymidine or BrdU), stem cell antigen Sca-1 expression, ability to exclude dyes such as Hoechst 33342 or rhodamine 123 (side population -- SP, e.g. Hoechst 33342 negative) because of elevated expression of membrane transporter proteins, such as P-glycoproteins, and “small light” cell by electronic microscopy. However, these profiling methods are controversial and confusing. Sometimes, for a layman, it is very difficult to handle. But this situation is about to change with two recent publications in Nature. In January, Shackleton et al published “Generation of a functional mammary gland from a single stem cell” 4. In this report, the authors cleared mammary gland mixture with CD31 (endothelial marker), CD45 and TER119 (haematopoietic antigens) by FACS sorting (Lin- population). Using repopulating cleared mammary fat pad (Mammary repopulating 'units'--MRUs) as criteria, they were able to increase the MRUs from 1/4900 to 1/64 just by applying two more markers on Lin- population -- CD29 (beta1-integrin) and CD24 (heat-stable antigen). Lin-CD29hiCd24+ cells have expended differentiation ability and colony-formation ability. A single Lin-CD29hiCD24+ cell can repopulate cleared mammary fat pad and develop into a fully functioning mammary gland, demonstrating its high proliferating and multi-lineage differentiation capacity in vivo. Lin-CD29hiCd24+ cells can self-renew. In mammary gland of MMTV-Wnt-1 transgenic mice, Lin-CD29hiCd24+ population are increased and mammary gland outgrowths of Lin-CD29hiCd24+ MMTV-Wnt-1 cells are profoundly hyperplastic. Lin-CD29hiCd24+ cells were enriched for long-term label-retaining cells, CD49f+ cells. However, neither high Sca-1 expression nor Hoechst33342 dye exclusion was enriched in this population. In the February publication “Purification and unique properties of mammary epithelial stem cells” 5, Stingl et al purified CD45-Ter119-CD31-CD49fhiCD24med cells and demonstrated that they were the mammary gland stem cells. In consistency, CD45-Ter119-CD31-CD49fhiCD24med cells were Sca-1 negative and only minority of these cells can efflux Hoechst 33342 and Rhodamine-123. Interestingly, the authors took one step further to illustrate that CD45-Ter119-CD31-CD49fhiCD24med cells are in G1 or S/G2/M fractions, indicating the stem cell population is a cycling population. Most notably, these two publications completely changed the old mammary gland stem cell picture -- Hoechst 33342 negative, slowly dividing and Sca-1 positive. They demonstrated that CD45-Ter119-CD31-CD49fhiCD24med and Lin-CD29hiCd24+ are the mammary stem cell populations, whereas previous SP and Sca-1+ cells only take very few percentage of these two populations if not at all. Since label retention coincides very well with Lin-CD29highCD24+ or CD45-Ter119-CD31-CD49fhiCD24med, it joins CD29, CD49f and CD24 as one of the most efficient 4 mammary gland stem cell markers. These new markers make it easier to isolate mammary gland stem cells, therefore open a door for further characterizing these cells. Importantly, with the same markers, cancer stem cells can be purified as well. This provides a new opportunity to develop new targeted therapies to killing cancer stem cells. Finally, the report proved that mammary gland stem cells were actually cycling within cell cycle. This observation lays an important foundation for testing new methods of chemoprevention and chemotherapy.

In a case-control study of 1868 breast cancer patients and 3391 control patients we searched for characteristics that predicted risk of breast cancer diagnosed before and after menopause. Common to increased risk of this disease in both periods of womanhood were: early menarche and late menopause; delayed marriage and first childbirth; more nulliparity or reduced gravidity and parity; reduced frequency of abortions; shorter overall child-bearing interval; more advanced education, higher socioeconomic status, and more contraceptive usage; and familial tendencies toward the disease. Breast cancer patients diagnosed before menopause were leaner than controls at age 20 and at time of diagnosis, but breast cancer risk in the postmenopausal period was related to increased weight-for-height at diagnosis and greater weight-for-height at diagnosis and greater weight gain since age 20. Postmenopausal breast cancer patients had a longer interval between first and second childbirths. Frequency and duration of the gravid state, inversely related to breast cancer risk, were largely dependent on contraceptive practices rather than unexplained infertility per se. Whether the breast cancer reaches diagnosis before or after menopause, the bulk of evidence examined here supports the view that it has a common cause and is subject to modifying influences over the long period of cancer latency.

Breast and colorectal cancers are among the most common tumor types worldwide and the occurrence of metastases is often associated with a shortened lifespan. One of the signaling pathways that has been frequently associated with metastasis in these tumor entities is the Wnt signaling pathway. Wnt signaling can be either β-catenin dependent (canonical) or βcatenin independent (non-canonical). In breast and colorectal cancer, tumor-promoting properties could be attributed to members of the non-canonical Wnt signaling pathway. Preliminary results showed that overexpression of ROR2 as a non-canonical Wnt receptor, mediated an aggressive phenotype in breast cancer cells and could significantly increase invasion. Accordingly, the first aim of this work was to investigate which ligand binds ROR2 and thus triggers the invasive behavior of MCF-7 cells. RNA-seq analysis revealed increased expression levels of the non-canonical ligand Wnt11 in ROR2 overexpressing cells. Hence, Wnt11 was further investigated as a potential ROR2-ligand. Using co-immunoprecipitation experiments, this work demonstrated the interaction of ROR2 with Wnt11 in human breast cancer cells. To determine which domain facilitates the ROR2-mediated invasion, sequential deletions of the different ROR2 domains were induced. This demonstrated that the cysteine-rich and the tyrosine kinase domain mediate this effect. The next step was to determine whether Wnt11 binds other receptors that trigger the invasive behavior of MCF-7 cells since ROR2 is not expressed in these cells. Furthermore, a cell line screening of different breast and colorectal cancer cell lines identified FZD4 and FZD6 as highly expressed non-canonical Wnt receptors. An interaction between Wnt11 and FZD6 was validated by using Co-IP, in which PTK7 appears to act as a co-receptor. To investigate functional implications of Wnt11-mediated signaling in breast cancer, MCF-7 cells were stimulated with recombinant Wnt11, which caused increased invasion and migration rates, whereas loss of FZD6 resulted in a significant decrease of the elevated invasion rates. In line with the collected data, a signature with non-canonical Wnt pathway members including FZD receptors, ROR receptors, Wnt ligands and PI3K signaling members was defined and was analyzed for its DMFS prognostic values in breast and colorectal cancer patients. Therefore, the signature was applied to gene expression data of primary breast and colorectal cancer patients. For the primary breast cancer patients, the signature clustered the data set into two patient groups, among which the group with high Wnt11 expression was associated with poor DMFS. Considering FZD4 and FZD6 individually, breast cancer patients with high FZD6 gene expression showed worse DMFS, while high FZD4 levels were associated with favorable DMFS. Interestingly, the defined signature clustered the data set of the colorectal cancer patients into four groups and the cohort with a high FZD6 expression exhibited a poor DMFS compared to the other groups. Another important aspect of tumor progression is the reprogramming of the tumor microenvironment. It has been shown that tumor-derived extracellular vesicles can influence the tumor microenvironment in different ways. Preliminary data demonstrated that Wnt proteins can be transported via extracellular vesicles to target cells and induce Wnt signaling responses there. It was shown that RORs are transported on microvesicles and exosomes. Modulation of ROR1 and ROR2 expression resulted in altered protein compositions for both vesicle populations. However, no major impact on vesicle size or concentration was evident. Further analysis addressed functional consequences of ROR1 and ROR2 expression on extracellular vesicles. Tumor-derived EVs isolated from the aggressive breast cancer cell line MDA-MB231 induced invasiveness in MCF-7 cells, which was shown to be dependent on vesicular ROR1 expression. To determine whether ROR1/2 can function as a biomarker in breast cancer patients, plasma-derived microvesicles were analyzed for their ROR1 and ROR2 expression by flow cytometry, which is currently ongoing. In conclusion, this work demonstrated the importance of non-canonical Wnt signaling in breast cancer progression. Wnt11 has been identified as a novel ligand for ROR2 and FZD6 by co-immunoprecipitation experiments, thereby mediating tumor-promoting properties in breast cancer. In particular, it has been shown that ROR proteins not only play an important role in breast cancer cells themselves, but also appear to be additionally involved in vesicle biogenesis and can furthermore be transferred to target cells. A clinical applicability of both ROR proteins regarding their usage as tumor biomarkers for breast cancer is still under investigation.

Background: The ovarian hormones estrogen and progesterone profoundly influence breast cancer risk, underpinning the benefit of endocrine therapies in the treatment of breast cancer. Modulation of their effects through ovarian ablation or chemoprevention strategies also significantly decreases breast cancer incidence. Conversely, there is an increased risk of breast cancer associated with pregnancy in the short-term. The cellular mechanisms underlying these observations, however, are poorly defined. We and others recently isolated mammary epithelial populations enriched for mammary stem cells (MaSCs), committed luminal progenitor and mature luminal cells from both mouse and human mammary glands. Unexpectedly, MaSCs exhibited a receptor-negative phenotype for ERα , PR and ErbB2. Given the central important of estrogen and progesterone signaling to mammary gland development and cancer, we sought to determine whether these hormones could indirectly modulate MaSC function. Methods and Results: We utilized mouse models to directly study the effects of steroid hormones on the in vivo repopulating ability of MaSCs. Ovariectomy markedly diminished MaSC number and the extent of ductal outgrowth in vivo. The relative contribution of estrogen and progesterone to the regulation of MaSC activity was next examined using hormone pellets or antagonists. MaSC activity increased in animals treated with both estrogen and progesterone. Remarkably, even three weeks of treatment with the aromatase inhibitor letrozole was sufficient to reduce the MaSC pool. The outgrowth potential of these cells was again affected, suggesting that MaSCs retain a ‘memory’ of estrogen deprivation, perhaps through perturbation of their cycling status. Indeed, cell cycle analysis revealed an increase in the percentage of MaSC-enriched cells in G0/G1 in ovariectomized glands compared to controls. This was accompanied by a profound reduction in the expression of cell cycle genes including Cyclin D1. We further evaluated the effect of the hormonal environment on MaSC function during pregnancy, where progesterone (and prolactin) have prominent roles. Pregnancy led to a transient 11-fold increase in MaSC numbers. This was accompanied by marked elevation in the expression of the progesterone target gene RANK ligand in luminal cells, together with its receptor RANK in the MaSC-containing population. To determine whether MaSC activity is in part mediated through paracrine signals from RANK ligand, inhibitors of RANK signaling were evaluated. Treatment of virgin or pregnant mice with an anti-RANK ligand monoclonal antibody in vivo significantly impaired the clonogenic activity of the MaSC-enriched but not luminal subpopulation. Discussion: Despite lacking the steroid hormone receptors ERα and PR, MaSCs appear to be exquisitely sensitive to hormone signaling, presumably via paracrine signaling that includes the RANK signaling pathway. The augmented MaSC pool during pregnancy suggests a cellular basis for the short-term increase in breast cancer incidence following pregnancy. Our findings further indicate that breast cancer chemoprevention may in part be achieved through suppression of MaSC function. We speculate that inhibitors of RANK and other stem cell signaling pathways could represent potential chemoprevention agents. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr S5-6.

Breast cancer risk and hormone replacement therapy among BRCA carriers after risk-reducing salpingo-oophorectomy

Mammary stem cells (MSCs) are the progenitor population for human breast epithelia. MSCs give rise during mammary gland development to estrogen receptor (ER)-negative basal cells and the ER- luminal progenitor (LP) population which maintains ER+ and ER- luminal cells. The MSC population is expanded and tumorigenic in some mouse mammary cancer models, and these tumor-initiating cells have been isolated from human breast cancers. MSC expansion is associated with aggressive biological behavior in human breast cancer. The LP population is tumorigenic in some mouse mammary cancer models, and is the progenitor population of basal breast cancer in humans. The enhancer of zeste homolog 2 (EZH2) is a methyltransferase which catalyzes lysine 27 methylation in histone H3 resulting in suppression of target gene expression. The histone demethylase JMJD3 opposes the activity of EZH2 by demethylating histone H3 lysine 27. EZH2 is a member of the polycomb group of proteins which regulates cell type identity. EZH2 expression was found to be increased in histologically normal human breast tissue among women with high breast cancer risk, and was elevated in ductal hyperplasia and ductal carcinoma in situ. EZH2 overexpression is associated with poorly differentiated and aggressive breast cancer in humans. However, the mechanisms by which EZH2 results in increased breast cancer risk and aggressive tumors are not completely characterized. Using in vivo transplantation of mammary cancer stem cells transduced with EZH2 or JMJD3 shRNAs, we demonstrated that EZH2 promotes mammary stem and LP cell expansion, metastasis and inhibits ER-positive cellular differentiation.

Background: Breast cancer, a leading malignancy in women, is influenced by hormonal and reproductive factors. Early menarche and late menopause increase breast cancer risk due to prolonged estrogen exposure. Objective: To investigate associations between reproductive factors (early menarche, late menopause) and breast cancer risk. Methods: This cross-sectional study at Dhaka Medical College (June 2023 to May 2024) included 32 breast cancer patients. Data were collected via questionnaires and medical records. Statistical analysis was performed using SPSS version 26. Ethical approval and informed consent were obtained. Results: The study included 32 participants with a mean age of 45.2 ± 10.5 years. Early menarche (≤11 years) was observed in 28.1%, and 56.2% were postmenopausal. A positive family history was reported in 37.5%, with 58.3% having an affected mother. Ductal carcinoma (75.0%) was the most common histological type, and 62.5% were ER-positive. Most cases were diagnosed at Stage II (37.5%) or Stage III (28.1%), emphasizing late detection. Conclusion: This study underscores the role of reproductive factors and family history in breast cancer risk, emphasizing the need for early screening. Mediscope 2026;13(1): 16-21

<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>

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.