Anchorage-independent Growth Of MCF-7 Cells Research Articles

Decreasing O-GlcNAcylation affects the malignant transformation of MCF-7 cells via Hsp27 expression and its O-GlcNAc modification.

O-GlcNAcylation is a dynamic posttranslational modification of nucleoplasmic proteins. Previously, we reported that the O-GlcNAcylation level was increased in primary breast and colorectal cancer tissues. However, its precise roles in cancer development and progression are still largely unexplored. The aim of the present study was to investigate the roles of O-GlcNAcylation in the malignant transformation of cancer cell lines. O-GlcNAcylation level was examined in six cancer cell lines including breast (MCF-7 and MDA-MB-231), colorectal (SW480 and SW620), and liver (SK-Hep1 and HepG2). We found that the levels of O-GlcNAcylation and O-GlcNAc transferase (OGT), an O-GlcNAc catalyzing enzyme, were obviously increased in all cancerous cells, except SK-Hep1, when compared to normal cells. Reducing O-GlcNAcylation using RNA interference against OGT showed a marked reduction in OGT and O-GlcNAcylation levels. Surprisingly, siOGT had no effect on cell growth under conventional monolayer cultures. However, it inhibited anchorage-independent growth in soft agar cultures of all cancer cells, except SK-Hep1. Under anoikis resistance conditions performed by spheroid cultures, siOGT treatment decreased viability only in MCF-7, SW480, and SW620 cells. Among them, OGT knockdown in MCF-7 cells revealed a high inhibitory effect on colony and spheroid cultures. Using two-dimensional gel electrophoresis and mass spectrometric analysis, heat shock protein 27 (Hsp27) was found to be the highest upregulated protein upon OGT knockdown. Immunoblots revealed that the Hsp27 protein level was increased but its O-GlcNAc modification level was decreased in siOGT-treated cells. These changes were associated with the inhibition of MCF-7 cell transformation. Notably, double knockdown of OGT and Hsp27 showed a reversal in the inhibitory effect on colony and spheroid cultures. Collectively, these results indicate that O-GlcNAcylation is required for anoikis resistance and anchorage-independent growth of MCF-7 cells. Blocking this glycosylation by OGT knockdown may regulate both Hsp27 protein expression and its O-GlcNAc modification levels. This alteration may play vital roles in malignant transformation.

Polymer Nanovesicle-Mediated Delivery of MLN8237 Preferentially Inhibits Aurora Kinase A To Target RalA and Anchorage-Independent Growth in Breast Cancer Cells.

The small GTPase RalA is a known mediator of anchorage-independent growth in cancers and is differentially regulated by adhesion and aurora kinase A (AURKA). Hence, inhibiting AURKA offers a means of specifically targeting RalA (over RalB) in cancer cells. MLN8237 (alisertib) is a known inhibitor of aurora kinases; its specificity for AURKA, however, is compromised by its poor solubility and transport across the cell membrane. A polymer nanovesicle platform is used for the first time to deliver and differentially inhibit AURKA in cancer cells. For this purpose, polysaccharide nanovesicles made from amphiphilic dextran were used as nanocarriers to successfully administer MLN8237 (VMLN) in cancer cells in 2D and 3D microenvironments. These nanovesicles (<200 nm) carry the drug in their intermembrane space with up to 85% of it released by the action of esterase enzyme(s). Lysotracker experiments reveal the polymer nanovesicles localize in the lysosomal compartment of the cell, where they are enzymatically targeted and MLN released in a controlled manner. Rhodamine B fluorophore trapped in the nanovesicles hydrophilic core (VMLN+RhB) allows us to visualize its uptake and localization in cells in a 2D and 3D microenvironment. In breast cancer, MCF-7 cells VMLN inhibits AURKA significantly better than the free drug at low concentrations (0.02-0.04 μM). This ensures that the drug in VMLN at these concentrations can specifically inhibit up to 94% of endogenous AURKA without affecting AURKB. This targeting of AURKA causes the downstream differential inhibition of active RalA (but not RalB). Free MLN8237 at similar concentrations and conditions failed to affect RalA activation. VMLN-mediated inhibition of RalA, in turn, disrupts the anchorage-independent growth of MCF-7 cells supporting a role for the AURKA-RalA crosstalk in mediating the same. These studies not only identify the polysaccharide nanovesicle to be an improved way to efficiently deliver low concentrations of MLN8237 to inhibit AURKA but, in doing so, also help reveal a role for AURKA and its crosstalk with RalA in anchorage-independent growth of MCF-7 cells.

Abstract 3121: Validation of protein kinase D1 as a prognostic factor and pharmacological target for the treatment of breast cancer

Abstract Tamoxifen-resistant ERα positive (ERα+) breast cancers and triple-negative breast cancers represent two aggressive subgroups of mammary tumors with limited treatment options and a poor prognosis. Thus, there is still an urgent need to develop and improve targeted pharmacological treatments against these estrogen-independent cancers. Recent data from our group suggest that the protein kinase D1 (PKD1) participates in the development and/or maintenance of the estrogen-independent phenotype in breast cancer. Indeed, up-regulating PKD1 conferred anchorage-independent growth to estrogen-dependent MCF-7 cells in the absence of β-estradiol. In addition, we found that high PKD1 mRNA levels were associated with a worse prognosis in tamoxifen-treated tumors. PKD1 is a serine-threonine kinase encoded by the prkd1 gene and belongs to the protein kinase D (PKD) family, a novel subgroup of the calcium and calmodulin-regulated kinases (CAMK). The PKD family also includes PKD2 and PKD3 which share a high homology with PKD1. The objective of the present study was to determine whether PKD1 could be a prognostic factor and/or a pharmacological target for the treatment of breast cancer. Expression of prkd1, prkd2 and prkd3 was analyzed by RT-qPCR in 527 breast cancer specimens (181 ERα- tumors including 102 triple negative tumors and 346 ERα+ tumors). We showed that higher prkd1 mRNA levels were associated with a lower metastasis-free survival in the overall breast cancer population (p=0.015). prkd1 prognostic value was even stronger in ERα- tumors (p=0.00042) and in triple negative tumors (p=0.0043). Conversely, prkd1 expression was not associated with prognosis in ERα+ tumors. Interestingly, high prkd2 and prkd3 mRNA levels were associated with a better prognosis in the entire cohort (p=0.0074 and p=0.028 respectively). We have developed PKD1 inhibitors in collaboration with the AB Science pharmaceutical company and screened the ability of thirty-five compounds to inhibit anchorage-independent growth of MCF-7 cells in the absence of estrogens. The antitumor activity of two PKD1 inhibitors is currently being evaluated in vivo against patient-derived breast cancer xenografts. In conclusion, our results show that high prkd1 expression is a bad-prognosis factor in breast cancer and that PKD1 could be a relevant therapeutic target for the treatment of breast cancer, in particular those harboring an estrogen-independent phenotype. Citation Format: Caroline Spasojevic, Marie Regairaz, Sophie Vacher, Franck Assayag, Jean Marc Ricort, Elisabetta Marangoni, Ivan Bièche, Christian Auclair. Validation of protein kinase D1 as a prognostic factor and pharmacological target for the treatment of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3121. doi:10.1158/1538-7445.AM2017-3121

Abstract 2446: Signaling event of alcohol-induced deregulation of Pol III genes in breast cancer cells

Abstract Alcohol intake is consistently associated with an increased risk of breast cancer. Alcohol consumption is more pronounced in ER+ breast cancer cases than in ER- cases. However, its mechanism remains to be determined. We reported that alcohol induces Pol III gene (RNA polymerase III-dependent genes) transcription in vivo and in vitro. We continue to define novel and unexpected targets, MSK1 (mitogen- stress-activated protein kinase 1) and Brf1 (TFIIIB-related factor 1), in the alcohol-induced response. Our aim is to delineate the mechanisms regulating the processes of deregulation of Pol III genes, cell transformation and tumorgenesis through this alcohol-induced response. Brf1 specifically regulates Pol III gene transcription. Changes in Pol III gene and Brf1 expression tightly link to cell transformation and tumor formation. Alcohol-induced deregulation of Pol III genes may be fundamental to the development of breast cancer. We have reported that a) enhancement of Brf1 and Pol III gene expression is correlated with tumor formation in alcohol-fed mice; b) alcohol increases ERα activity to elevate Brf1 and Pol III gene expression; c) alcohol activates JNK1 to increase ERα and Brf1 expression; d) ethanol induces H3ph (histone H3 phosphorylation), e) H3ph elevates Brf1 expression. However, nothing is known about the role of MSK1 in transcription of Brf1 and Pol III genes. Analysis of signal event has revealed that ethanol markedly stimulates phosphorylation of MSK1 at serine 376 and threonine 581 in MCF-7 cells. Blocking MSK1 singling decreases cellular level of Brf1 expression and Pol III gene transcription. Further analysis indicates that MSK1 mediates ERα expression. Inhibition of MSK signaling decreases alcohol-induced ERE-Luc reporter activity. Repression of ERα expression by its siRNA reduces alcohol-induced anchorage-independent growth of MCF-7 cells. Our results have demonstrated that repression of MSK1 by its siRNA decreases colony formation of MCF-7 cells. Further analysis of ChIP assay indicates that ethanol treatment increases occupancy of ERα to Brf1 promoter. These results support the idea that alcohol activates MSK1 and enhances ERα and Brf1 expression resulting greater phenotypic changes. Together, our studies indicate that ethanol-induced MSK1 activation may play a critical role in alcohol-induced cell transformation and alcohol-associated ER+ breast cancer. *: The project is supported by NIH grants AA017288 and AA021114 to Shuping Zhong Citation Format: Shuping Zhong, Qingsong Zhang, Ganggang Shi. Signaling event of alcohol-induced deregulation of Pol III genes in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2446. doi:10.1158/1538-7445.AM2014-2446

The SMRT coregulator enhances growth of estrogen receptor-α-positive breast cancer cells by promotion of cell cycle progression and inhibition of apoptosis.

The SMRT coregulator functions as a dual coactivator and corepressor for estrogen receptor-α (ERα) in a gene-specific manner, and in several studies its elevated expression correlates with poor outcome for breast cancer patients. A specific role of SMRT in breast cancer progression has not been elucidated, but SMRT knock-down limits estradiol-dependent growth of MCF-7 breast cancer cells. In this study, small-interfering RNA (siRNA) and short-hairpin RNA (shRNA) approaches were used to determine the effects of SMRT depletion on growth of ERα-positive MCF-7 and ZR-75-1 breast cancer cells, as well as the ERα-negative MDA-MB-231 breast cancer line. Depletion of SMRT inhibited growth of ERα-positive cells grown in monolayer but had no effect on growth of the ERα-negative cells. Reduced SMRT levels also negatively impacted the anchorage-independent growth of MCF-7 cells as assessed by soft agar colony formation assays. The observed growth inhibitions were due to a loss of estradiol-induced progression through the G1/S transition of the cell cycle and increased apoptosis in SMRT-depleted compared with control cells. Gene expression analyses indicated that SMRT inhibits apoptosis by a coordinated regulation of genes involved in apoptosis. Functioning as a dual coactivator for anti-apoptotic genes and corepressor for pro-apoptotic genes, SMRT can limit apoptosis. Together these data indicate that SMRT promotes breast cancer progression through multiple pathways leading to increased proliferation and decreased apoptosis.

Correlation between PDZK1, Cdc37, Akt and breast cancer malignancy: the role of PDZK1 in cell growth through Akt stabilization by increasing and interacting with Cdc37.

PDZ domain containing 1 (PDZK1) is a scaffold protein that plays a role in the fate of several proteins. Estrogen can induce PDZK1 gene expression; however, our recent report showed that PDZK1 expression in the breast cancer cell line MCF-7 is indirect and involves insulin-like growth factor (IGF)-1 receptor function. Such a relationship was established in cell culture systems and human breast cancer tissues. Here we show that overexpression of PDZK1 promoted an increase in cyclin D1 and enhanced anchorage-independent growth of MCF-7 cells in the absence of 17β-estradiol, suggesting that PDZK1 harbors oncogenic activity. Indeed, PDKZ1 overexpression enhanced epidermal growth factor receptor (EGFR)-stimulated MEK/ERK1/2 signaling and IGF-induced Akt phosphorylation. PDZK1 appeared to play this role, in part, by stabilizing the integrity of the growth promoting factors Akt, human epidermal growth factor receptor 2 (Her2/Neu) and EGFR. Increased Akt levels occurred via a decrease in the ubiquitination of the kinase. PDZK1 overexpression was associated with resistance to paclitaxel/5-fluorouracil/etoposide only at low concentrations. Although the increased stability of Akt was sensitive to heat shock protein 90 (HSP90) inhibition, increased levels of the cochaperone cell division cycle 37 (Cdc37), as well as its ability to bind PDZK1, appear to play a larger role in kinase stability. Using human tissue microarrays, we show strong positive correlation between PDZK1, Akt and Cdc37 protein levels, and all correlated with human breast malignancy. There were no positive correlations between PDZK1 and Cdc37 at the mRNA levels, confirming our in vitro studies. These results demonstrate a relationship between PDZK1, Akt and Cdc37, and potentially Her2/Neu and EGFR, in breast cancer, representing a new axis that can be targeted therapeutically to reduce the burden of human breast cancer.

Abstract LB-5: The dominant-negative effect of an insulin-like growth factor-1 mutant that is defective in binding to integrins (αvβ3 and α6β4)

Abstract Insulin-like growth factor-1 (IGF1) is a major therapeutic target for cancer. Integrin αvβ3 plays a critical role in regulating IGF1 signaling, while the specifics are unclear. We recently reported that IGF1 directly binds to αvβ3. The integrin-binding defective IGF1 mutant (R36E/R37E) is defective in inducing IGF signaling and a ternary complex (αvβ3, IGF1R, and IGF1) formation, although the mutant still binds to the type 1 IGF receptor (IGF1R). These findings suggest that direct binding of IGF1 to αvβ3 is required for intracellular signaling (J. Saegusa, S. Yamaji, K. Ieguchi, C.Y. Wu, K.S. Lam, F.T. Liu, Y.K. Takada, and Y. Takada, The direct binding of insulin-like growth factor-1 (IGF-1) to integrin αvβ3 is involved in IGF-1 signaling. J Biol Chem, 2009. 284: 24106–14). Here we describe that R36E/R37E suppressed proliferation and intracellular signaling induced by WT IGF1 in NIH3T3 fibroblasts, C2C12 myoblasts, and MCF-7 breast cancer cells (a dominant negative effect). R36E/R37E suppressed anchorage-independent growth of MCF-7 cells, while WT IGF1 enhanced it. Also, R36E/R37E suppressed the tumorigenesis of MCF-7 cells in SCID mice, while wt IGF-1 markedly enhanced it. Since MCF-7 cells express little or no αvβ3, we studied which integrins are involved in IGF signaling. We discovered that α6β4, which is over-expressed in cancer and implicated in tumor progression, directly binds to WT IGF1 (but not to R36E/R37E) in MCF-7. Interestingly, WT IGF1 induced a ternary complex formation (α6β4, IGF1, and IGF1R) but R36E/R37E did not. These results suggest that α6β4 in addition to αvβ3 plays a role in IGF signaling through direct binding to IGF1, and that R36E/R37E acts as an inhibitor of IGF signaling. We propose that R36E/R37E has potential as a therapeutic agent in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-5. doi:10.1158/1538-7445.AM2011-LB-5

Signal Transducer and Activator of Transcription (STAT)-A and STAT5B Differentially Regulate Human Mammary Carcinoma Cell Behavior.

Abstract Increased signal transducer and activator of transcription (STAT)-5 activation has been reported in various malignancies including mammary carcinoma. However, it is only recently that potentially distinct roles of STAT5A and STAT5B in neoplasia have begun to emerge. Herein we systematically delineate the functions of STAT5A and STAT5B in human mammary carcinoma cells. Forced expression of constitutively active (CA) STAT5A enhanced both survival and anchorage-independent growth of human mammary carcinoma cells but concordantly suppressed cell motility as revealed in colony scattering, cell migration, and invasion assays. In contrast, forced expression of CA STAT5B exhibited lower potency than CA STAT5A in enhancing survival and anchorage-independent growth of mammary carcinoma cells and exerted no effects on cell motility. Differential expression of genes that regulate cellular survival and motility was concomitantly observed on forced expression of CA STAT5A or CA STAT5B. We also observed that functions of STAT5A and STAT5B were similarly differential in T47D cells. Small interfering RNA-mediated depletion of STAT5A significantly impaired anchorage-independent growth of MCF-7 cells, whereas a smaller reduction was observed upon small interfering RNA-mediated depletion of STAT5B. Depletion of endogenous STAT5A also significantly enhanced cell motility, whereas depletion of endogenous STAT5B exhibited no effect. Xenograft studies provided data concordant with the in vitro effects of the two STAT5 isoforms. We therefore demonstrate that STAT5A and STAT5B differentially regulate behavior of human mammary carcinoma cells.

Signal Transducer and Activator of Transcription (STAT)-A and STAT5B Differentially Regulate Human Mammary Carcinoma Cell Behavior

Increased signal transducer and activator of transcription (STAT)-5 activation has been reported in various malignancies including mammary carcinoma. However, it is only recently that potentially distinct roles of STAT5A and STAT5B in neoplasia have begun to emerge. Herein we systematically delineate the functions of STAT5A and STAT5B in human mammary carcinoma cells. Forced expression of constitutively active (CA) STAT5A enhanced both survival and anchorage-independent growth of human mammary carcinoma cells but concordantly suppressed cell motility as revealed in colony scattering, cell migration, and invasion assays. In contrast, forced expression of CA STAT5B exhibited lower potency than CA STAT5A in enhancing survival and anchorage-independent growth of mammary carcinoma cells and exerted no effects on cell motility. Differential expression of genes that regulate cellular survival and motility was concomitantly observed on forced expression of CA STAT5A or CA STAT5B. We also observed that functions of STAT5A and STAT5B were similarly differential in T47D cells. Small interfering RNA-mediated depletion of STAT5A significantly impaired anchorage-independent growth of MCF-7 cells, whereas a smaller reduction was observed upon small interfering RNA-mediated depletion of STAT5B. Depletion of endogenous STAT5A also significantly enhanced cell motility, whereas depletion of endogenous STAT5B exhibited no effect. Xenograft studies provided data concordant with the in vitro effects of the two STAT5 isoforms. We therefore demonstrate that STAT5A and STAT5B differentially regulate behavior of human mammary carcinoma cells.

Period-2: a tumor suppressor gene in breast cancer

Previous reports have suggested that the ablation of the Period 2 gene (Per 2) leads to enhanced development of lymphoma and leukemia in mice. Employing immunoblot analyses, we have demonstrated that PER 2 is endogenously expressed in human breast epithelial cell lines but is not expressed or is expressed at significantly reduced level in human breast cancer cell lines. Expression of PER 2 in MCF-7 breast cancer cells significantly inhibited the growth of MCF-7 human breast cancer cells, and, when PER 2 was co-expressed with the Crytochrome 2 (Cry 2) gene, an even greater growth-inhibitory effect was observed. The inhibitory effect of PER 2 on breast cancer cells was also demonstrated by its suppression of the anchorage-independent growth of MCF-7 cells as evidenced by the reduced number and size of colonies. A corresponding blockade of MCF-7 cells in the G1 phase of the cell cycle was also observed in response to the expression of PER 2 alone or in combination with CRY 2. Expression of PER 2 also induced apoptosis of MCF-7 breast cancer cells as demonstrated by an increase in PARP [poly (ADP-ribose) polymerase] cleavage. Finally, our studies demonstrate that PER 2 expression in MCF-7 breast cancer cells is associated with a significant decrease in the expression of cyclin D1 and an up-regulation of p53 levels.

IQGAP1 Stimulates Proliferation and Enhances Tumorigenesis of Human Breast Epithelial Cells

The scaffold protein IQGAP1 integrates signaling pathways and participates in diverse cellular activities. IQGAP1 is overexpressed in a number of human solid neoplasms, but its functional role in tumorigenesis has not been previously evaluated. Here we report that IQGAP1 contributes to neoplastic transformation of human breast epithelial cells. The amount of IQGAP1 in breast carcinoma is greater than that in normal tissue, with highly metastatic breast epithelial cells expressing the highest levels. Overexpression of IQGAP1 enhances proliferation of MCF-7 breast epithelial cells. Reduction of endogenous IQGAP1 by RNA interference impairs both serum-dependent and anchorage-independent growth of MCF-7 cells. Consistent with these in vitro observations, immortalized MCF-7 cells overexpressing IQGAP1 form invasive tumors in immunocompromised mice, whereas tumors derived from MCF-7 cells with stable knockdown of IQGAP1 are smaller and less invasive. In vitro analysis with selected IQGAP1 mutant constructs and a chemical inhibitor suggests that actin, Cdc42/Rac1, and the mitogen-activated protein kinase pathway contribute to the mechanism by which IQGAP1 increases cell invasion. Collectively, our data reveal that IQGAP1 enhances mammary tumorigenesis, suggesting that it may be a target for therapeutic intervention.

Solution Structure and Antiestrogenic Activity of the Unique C-terminal, NR-box Motif-containing Region of MTA1s

Metastasis tumor-associated 1 short form (MTA1s) is a naturally occurring, alternatively spliced variant of MTA1 that functions as a repressor of estrogen receptor (ER) alpha transcriptional functions, at least in part by binding and sequestering ERalpha in the cytoplasm. A unique C-terminal 33-amino acid region containing a nuclear receptor (NR)-box motif (-LRILL-) mediates binding of MTA1s with ERalpha and is indispensable in this interaction. Here, we elucidated the solution structure of this 33-amino acid region by NMR spectroscopy. We found a predominance of the alpha-helical region toward the N-terminal region, which includes the NR-box motif. In silico docking and comparison studies showed similarities between the NR-box motif of MTA1s and a similar motif of coregulators, both in structure and mode of ERalpha binding. In MCF-7 breast cancer cells, the MTA1s peptide effectively repressed ERalpha transactivation function, as evidenced by the estrogen response element-luc assay and down-regulation of estrogen-induced genes. In mechanistic studies, we found that the antiestrogenic effects of the MTA1s peptide were due to its ability to compete with the coactivator recruitment to ERalpha. Furthermore, the peptide efficiently repressed estrogen-induced proliferation and anchorage-independent growth of MCF-7 cells. In addition, the MTA1s peptide blocked the progression of tumors formed by MCF-7 cells overexpressing an ERalpha coactivator in a xenograft-based assay. In brief, the characterization of structure and antiestrogenic activity of MTA1s peptide highlight its therapeutic potential.

An inhibitory function for JNK in the regulation of IGF-I signaling in breast cancer.

Insulin-like growth factor-I receptor (IGF-IR) is frequently overexpressed in a variety of cancer types. Since many breast tumors and cancer cell lines overexpress IGF-IR, we tested IGF-I effects on chemotherapy-treated breast cancer cells. IGF-I protects from chemotherapy-induced apoptosis, suggesting that overlapping signaling pathways modulate IGF-I and chemotherapy treatment outcomes. Taxol and other chemotherapy drugs induce c-Jun N-terminal kinase (JNK), a kinase that conveys cellular stress and death signals. Notably, in this paper we show that IGF-I alone induces a potent JNK response and this activity is reversed by inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) with LY294002 in MCF-7 but not T47D cells. Cotreatment of cells with chemotherapy and IGF-I leads to additive JNK responses. Using cells overexpressing Akt, we confirm that IGF-I-mediated survival is Akt dependent. In contrast, overexpression of JNK significantly enhances Taxol-induced apoptosis and inhibits IGF-I survival effects. Further, JNK attenuates anchorage-independent growth of MCF-7 cells. The inhibitory effect of JNK appears to be mediated by serine phosphorylation of IRS-1 (insulin receptor substrate) since both Taxol and IGF-I treatment enhanced Ser(312) IRS-1 phosphorylation, while LY294002 blocked IGF-I-mediated phosphorylation. Taken together, these data provide a mechanism whereby stress or growth factors activate JNK to reduce proliferation and/or survival in breast cancer cells.

LATS1 tumor suppressor regulates G2/M transition and apoptosis.

The LATS1 gene is a mammalian member of the novel lats tumor suppressor family. Both lats mosaic flies and LATS1 deficient mice spontaneously develop tumors. Our previous studies have shown that inactivation of Drosophila lats leads to up-regulation of cyclin A in the fly, and the human LATS1 protein associates with CDC2 in early mitosis in HeLa cells, suggesting that the lats gene family may negatively regulate cell proliferation by modulating CDC2/Cyclin A activity. We demonstrate here that transduction of the human breast cancer cell MCF-7 with recombinant LATS1 adenovirus (Ad-LATS1), but not with EGFP adenovirus (Ad-EGFP), inhibits in vitro cell proliferation. Ectopic expression of LATS1 in MCF-7 cells specifically down-regulates Cyclin A and Cyclin B protein levels and dramatically reduces CDC2 kinase activity, leading to a G2/M blockade. Furthermore, Ad-LATS1 suppresses anchorage-independent growth of MCF-7 cells in soft agar and tumor formation in athymic nude mice. We also demonstrate that ectopic expression of LATS1 in MCF-7 cells and human lung cancer cell H460 up-regulates the level of BAX proteins and induces apoptosis. Finally, we show that LATS1 kinase activity is required for its ability to inhibit cell growth and induce apoptosis. The results indicate that the LATS1 tumor suppressor may play an important role in the control of human tumor development and that LATS1 suppresses tumorigenesis by negatively regulating cell proliferation and modulating cell survival.

Alpha 1-antitrypsin blocks the release of transforming growth factor-alpha from MCF-7 human breast cancer cells.

Human breast cancer cells synthesize and release a variety of growth-modulating substances in response to estrogen stimulation, and it is generally accepted that the growth-promoting effects of estrogens are due at least in part to this autocrine/paracrine mechanism. Several of these growth-modulating substances, including transforming growth factor-alpha (TGF alpha) and its analogs, have been shown to require pericellular proteolysis for activation or release. Recently, we reported that MCF-7 human breast cancer cells are able to synthesize alpha 1-antitrypsin (alpha 1-AT), the major elastase inhibitor in human serum, and that there is a negative correlation between anchorage-independent growth of MCF-7 cells in soft agar and synthesis of alpha 1-AT. The studies we present here were undertaken to gain an understanding of the mechanisms responsible for this observation. We show that release of TGF alpha from its membrane-bound precursor on MCF-7 cells is blocked by alpha 1-AT whether the cells were maintained in the presence or absence of estradiol and that there is a clear dose-response relationship between the alpha 1-AT concentration and both the release of TGF alpha and growth in soft agar. Consistent with this, TGF alpha release was increased in the presence of antibody to alpha 1-AT. In contrast, TGF alpha release and growth in soft agar were not blocked by peptide inhibitors specific for trypsin- or chymotrypsin-like enzymes. The alpha 1-AT concentration required for a half-maximal effect is lower for inhibition of TGF alpha release than it is for inhibition of colony formation (0.4 vs. 1.5 mumol/L). However, both values are in the range of concentrations one might expect at the cell surface in vivo. A new MCF-7 cell subline producing 10-fold higher levels of alpha 1-AT than its parent cell line was constructed by stable transfection of MCF-7 ML cells (a subline producing low levels of alpha 1-AT) with an alpha 1-AT complementary DNA. Growth in soft agar and release of TGF alpha were significantly decreased in cells transfected with the alpha 1-AT complementary DNA compared to those in cells transfected with vector alone, although, TGF alpha expression was the same. The above observations support a model for growth regulation in human breast ductal epithelial cells in which growth factor activation and release are dependent on the coordinate action of proteases and protease inhibitors. This model would predict that alpha 1-AT can act as a tumor suppressor in inhibiting the growth of breast cancer cells.

Alteratioon in proliferative and endocrine responsiveness of human mammary carcinoma cells by prototypic tumor-suppressing agents

The experiments performed in this study were designed to establish that (1) acquisition of anchorage-independent growth, a biological characteristic of tumorigenically transformed phenotype, can be modulated by prototypic tumor-suppressing agents, and (2) modulation of growth is influenced by the metabolic competence of the cells to biotransform estradiol. MCF-7 human breast carcinoma cells exhibited linear cell proliferative kinetics with a 41-hour population doubling time, and a 15% colony-forming efficiency in 0.33% agar. Indole-3-carbinol (I3C), a naturally occurring tumor-suppressive agent; tamoxifen (TAM), an antiestrogenic agent; and 4-hydroxytamoxifen (4-OHTAM), a metabolite of TAM, demonstrated 73.7%, 72.5%, and 89.9% suppression in anchorage-independent growth of MCF-7 cells, respectively. At the metabolic level, 13C and 4-OHTAM induced 2.3-fold ( P < 0.001) and 1.3-fold increase ( P = 0.001) relative to their own controls in the extent of 2-hydroxylation of estradiol. The results indicate that growth inhibition by I3C, TAM, and 4-OMTAM may in part be due to altered estradiol metabolism in MCF-7 cells. Thus, anchorage-independent growth and altered biotransformation of estradiol may constitute useful cellular and endocrine markers to evaluate the biological response of chemosuppressive agents.

Effects of the pineal hormone melatonin on the anchorage-independent growth of human breast cancer cells (MCF-7) in a clonogenic culture system

Only physiological levels of melatonin exert an antiproliferative effect on MCF-7 breast cancer cells grown in an anchorage-dependent culture system. We investigated melatonin's effect on the anchorage-independent growth of MCF-7 cells as well as the dose-response characteristics of this indoleamine under clonogenic culture conditions. Melatonin's inhibitory effect, with respect to the number and size of colonies formed, exhibit a linear dose-response curve with pharmacological concentrations producing a maximal inhibition while subphysiological levels of melatonin induce minimal inhibition. These results indicate that cellular attachment may modify the sensitivity of MCF-7 cells towards melatonin.