Doris Germain

Targeting the Cytoplasmic and Nuclear Functions of Signal Transducers and Activators of Transcription 3 for Cancer Therapy

Signal transducers and activators of transcription (STAT) are a highly conserved family of transcription factors that are activated by phosphorylation in the cytoplasm, after which they translocate to the nucleus to regulate gene expression. Among the seven STATs, STAT3 is of particular interest due to its constitutive phosphorylation in a large proportion of human cancers and its ability to induce neoplastic transformation. Inhibition of STAT3 can reverse tumor growth in experimental systems while having few effects in normal cells. These findings have implicated STAT3 as a potentially important target for therapeutic intervention. In addition to its well-described role as a transcription factor, STAT3 has been found recently to have important effects in the cytoplasm. Collectively, these functions of STAT3 directly contribute to tumorigenesis, invasion, and metastasis. Given the potential importance of STAT3 as a target for cancer therapy, molecules have been developed that can block STAT3 function at a variety of steps. These drugs show promise as anticancer agents in model systems of a variety of common human cancers. Thus, elucidating the functions of STAT3 and developing agents to inhibit this protein remain important scientific and clinical challenges.

Cyclin-dependent kinase inhibition by the KLF6 tumor suppressor protein through interaction with cyclin D1

Kruppel-like factor 6 (KLF6) is a tumor suppressor gene inactivated in prostate and colon cancers, as well as in astrocytic gliomas. Here, we establish that KLF6 mediates growth inhibition through an interaction with cyclin D1, leading to reduced phosphorylation of the retinoblastoma protein (Rb) at Ser795. Furthermore, introduction of KLF6 disrupts cyclin D1-cyclin-dependent kinase (cdk) 4 complexes and forces the redistribution of p21Cip/Kip onto cdk2, which promotes G1 cell cycle arrest. Our data suggest that KLF6 converges with the Rb pathway to inhibit cyclin D1/cdk4 activity, resulting in growth suppression.

SirT3 Regulates the Mitochondrial Unfolded Protein Response

ABSTRACT The mitochondria of cancer cells are characterized by elevated oxidative stress caused by reactive oxygen species (ROS). Such an elevation in ROS levels contributes to mitochondrial reprogramming and malignant transformation. However, high levels of ROS can cause irreversible damage to proteins, leading to their misfolding, mitochondrial stress, and ultimately cell death. Therefore, mechanisms to overcome mitochondrial stress are needed. The unfolded protein response (UPR) triggered by accumulation of misfolded proteins in the mitochondria (UPRmt) has been reported recently. So far, the UPRmt has been reported to involve the activation of CHOP and estrogen receptor alpha (ERα). The current study describes a novel role of the mitochondrial deacetylase SirT3 in the UPRmt. Our data reveal that SirT3 acts to orchestrate two pathways, the antioxidant machinery and mitophagy. Inhibition of SirT3 in cells undergoing proteotoxic stress severely impairs the mitochondrial network and results in cellular death. These observations suggest that SirT3 acts to sort moderately stressed from irreversibly damaged organelles. Since SirT3 is reported to act as a tumor suppressor during transformation, our findings reveal a dual role of SirT3. This novel role of SirT3 in established tumors represents an essential mechanism of adaptation of cancer cells to proteotoxic and mitochondrial stress.

Mitochondrial protein quality control by the proteasome involves ubiquitination and the protease Omi.

We report here that blocking the activity of the 26 S proteasome results in drastic changes in the morphology of the mitochondria and accumulation of intermembrane space (IMS) proteins. Using endonuclease G (endoG) as a model IMS protein, we found that accumulation of wild-type but to a greater extent mutant endoG leads to changes in the morphology of the mitochondria similar to those observed following proteasomal inhibition. Further, we show that wild-type but to a greater extent mutant endoG is a substrate for ubiquitination, suggesting the presence of a protein quality control. Conversely, we also report that wild-type but not mutant endoG is a substrate for the mitochondrial protease Omi but only upon inhibition of the proteasome. These findings suggest that although elimination of mutant IMS proteins is strictly dependent on ubiquitination, elimination of excess or spontaneously misfolded wild-type IMS proteins is monitored by ubiquitination and as a second checkpoint by Omi cleavage when the proteasome function is deficient. One implication of our finding is that in the context of attenuated proteasomal function, accumulation of IMS proteins would contribute to the collapse of the mitochondrial network such as that observed in neurodegenerative diseases. Another implication is that such collapse could be accelerated either by mutations in IMS proteins or by mutations in Omi itself.

Estrogen receptor mediates a distinct mitochondrial unfolded protein response

Unfolded protein responses (UPRs) of the endoplasmic reticulum and mitochondrial matrix have been described. Here, we show that the accumulation of proteins in the inter-membrane space (IMS) of mitochondria in the breast cancer cell line MCF-7 activates a distinct UPR. Upon IMS stress, overproduction of reactive oxygen species (ROS) and phosphorylation of AKT triggers estrogen receptor (ER) activity, which further upregulates the transcription of the mitochondrial regulator NRF1 and the IMS protease OMI (officially known as HTRA2). Moreover, we demonstrate that the IMS stress-induced UPR culminates in increased proteasome activity. Given our previous report on a proteasome- and OMI-dependent checkpoint that limits the import of IMS proteins, the findings presented in this study suggest that this newly discovered UPR acts as a cytoprotective response to overcome IMS stress.

Differential expression of the F-box proteins Skp2 and Skp2B in breast cancer

Skp2 is an F-box protein involved in the ubiquitination and subsequent degradation of the cyclin-dependent kinase (Cdk) inhibitor p27. Skp2 has been reported to be overexpressed in a variety of cancer types and to correlate with poor prognosis. We have identified a novel isoform of Skp2 we named Skp2B, which differs from Skp2 only in the C-terminal domain and unlike Skp2 localizes to the cytoplasm. Here, we describe the relative expression of both Skp2 and Skp2B in breast cancer cell lines and in primary breast cancers using quantitative real time RT–PCR. We show that Skp2B mRNA is expressed 10-fold less than Skp2 mRNA in the immortalized but non-transformed breast cell line, 184B5. However, Skp2B is overexpressed as frequently as Skp2, and to higher levels than Skp2 in breast cancer cell lines and primary cancers. Further, we show that cytoplasmic staining is frequent in primary breast cancers. In addition, we found that xenografts expressing Skp2B grow faster than xenografts expressing low levels of Skp2B, and that this effect is independent of p27 degradation. These findings therefore suggest that Skp2B overexpression is also observed in breast cancers and identify Skp2B as a putative oncogene.

A splice variant of Skp2 is retained in the cytoplasm and fails to direct cyclin D1 ubiquitination in the uterine cancer cell line SK-UT

Cyclin D1 is an important regulator of the transition from G1 into S phase of the cell cycle. The level to which cyclin D1 accumulates is tightly regulated. One mechanism contributing to the control of cyclin D1 levels is the regulation of its ubiquitination. SK-UT-1B cells are deficient in the degradation of D-type cyclins. We show here that p27, a substrate of the SCFSkp2 ubiquitin ligase complex, is coordinately stabilized in SK-UT-1B cells. Further, we show that expression of Skp2 in SK-UT-1B cells rescues the cyclin D1 and p27 degradation defect observed in this cell line. These results therefore indicate that the SCFSkp2 ubiquitin ligase complex affects the ubiquitination of cyclin D1. In addition, we show that SK-UT-1B cells express a novel splice variant of Skp2 that localizes to the cytoplasm and that cyclin D1 ubiquitination takes place in the nucleus. We propose that the translocation of Skp2 into the nucleus is required for the ubiquitination of cyclin D1 and that the absence of the SCFSkp2 complex in the nucleus of SK-UT-1B cells is the mechanism underlying the ubiquitination defect observed in this cell line. Finally, our data indicates that differential splicing of F-box proteins may represent an additional level of regulation of the F-box mediated ubiquitination pathway.

KLF6-SV1 Drives Breast Cancer Metastasis and Is Associated with Poor Survival

The KLF6-SV1 splice variant is associated with poor prognosis in early-stage human breast cancer and drives metastasis through the regulation of an EMT-like program in culture and in vivo. A New TWIST on Breast Cancer Sprawl Suburban sprawl is a complex, multistep process whereby “healthy” land becomes inundated with fast food chains, strip malls, and large parking lots. Yet, what drives sprawl is unclear—Do car-centric residential communities pop up close to businesses, or are the businesses merely opening where the consumers are? Cancer metastasis can be thought of as another type of sprawl, and although we can describe changes associated with metastasis, the drivers are equally unclear. Now, Hatami et al. provide insight into one of the potential drivers of breast cancer metastasis. The authors found that KLF6-SV1, which is a variant of a tumor suppressor gene, was associated with increased metastatic potential and poor survival in breast cancer patients. They then took their studies to the next step, trying to figure out how exactly KLF6-SV1 contributed to metastasis. Overexpressing KLF6-SV1 contributed to an epithelial-to-mesenchymal transition (EMT), which is thought to be important for cancer cells to leave the primary tumor. Indeed, inhibiting KLF6-SV1 returned these cells to a more epithelial (less metastatic) phenotype. Moreover, KLF6-SV1 alters the expression of TWIST1, which regulates EMT. Thus, KLF6-SV1 may be an early driver for metastasis in breast cancer patients. Metastasis is the major cause of cancer mortality. A more thorough understanding of the mechanisms driving this complex multistep process will aid in the identification and characterization of therapeutically targetable genetic drivers of disease progression. We demonstrate that KLF6-SV1, an oncogenic splice variant of the KLF6 tumor suppressor gene, is associated with increased metastatic potential and poor survival in a cohort of 671 lymph node–negative breast cancer patients. KLF6-SV1 overexpression in mammary epithelial cell lines resulted in an epithelial-to-mesenchymal–like transition and drove aggressive multiorgan metastatic disease in multiple in vivo models. Additionally, KLF6-SV1 loss-of-function studies demonstrated reversion to an epithelial and less invasive phenotype. Combined, these findings implicate KLF6-SV1 as a key driver of breast cancer metastasis that distinguishes between indolent and lethal early-stage disease and provides a potential therapeutic target for invasive breast cancer.

SOD2 to SOD1 Switch in Breast Cancer

Background: Cancer cells are characterized by elevated mitochondrial ROS. The dismutases SOD1 and SOD2 regulate ROS. Results: SOD2 is down-regulated following oncogenic activation in breast cancers. However, SOD1 is overexpressed, and its inhibition by LCS-1 leads to mitochondrial fragmentation. Conclusion: In the absence of SOD2, inhibition of SOD1 abolishes the integrity of the mitochondria. Significance: Our data suggest a SOD switch during transformation. Cancer cells are characterized by elevated levels of reactive oxygen species, which are produced mainly by the mitochondria. The dismutase SOD2 localizes in the matrix and is a major antioxidant. The activity of SOD2 is regulated by the deacetylase SIRT3. Recent studies indicated that SIRT3 is decreased in 87% of breast cancers, implying that the activity of SOD2 is compromised. The resulting elevation in reactive oxygen species was shown to be essential for the metabolic reprograming toward glycolysis. Here, we show that SOD2 itself is down-regulated in breast cancer cell lines. Further, activation of oncogenes, such as Ras, promotes the rapid down-regulation of SOD2. Because in the absence of SOD2, superoxide levels are elevated in the matrix, we reasoned that mechanisms must exist to retain low levels of superoxide in other cellular compartments especially in the intermembrane space of the mitochondrial to avoid irreversible damage. The dismutase SOD1 also acts as an antioxidant, but it localizes to the cytoplasm and the intermembrane space of the mitochondria. We report here that loss of SOD2 correlates with the overexpression of SOD1. Further, we show that mitochondrial SOD1 is the main dismutase activity in breast cancer cells but not in non-transformed cells. In addition, we show that the SOD1 inhibitor LCS-1 leads to a drastic fragmentation and swelling of the matrix, suggesting that in the absence of SOD2, SOD1 is required to maintain the integrity of the organelle. We propose that by analogy to the cadherin switch during epithelial-mesenchymal transition, cancer cells also undergo a SOD switch during transformation.

Tamoxifen Stimulates the Growth of Cyclin D1–Overexpressing Breast Cancer Cells by Promoting the Activation of Signal Transducer and Activator of Transcription 3

De novo or acquired resistance to tamoxifen is a major clinical challenge for the management of estrogen receptor (ER)-positive breast cancers. Although cyclin D1 overexpression is associated with a better outcome for breast cancer patients, its overexpression is also linked to tamoxifen resistance. We previously reported that the beneficial effect of cyclin D1 correlates with its ability to repress the antiapoptotic transcription factor signal transducer and activator of transcription 3 (STAT3). In contrast, molecular pathways linking overexpression of cyclin D1 to tamoxifen resistance have not been established. In the current study, the effect of tamoxifen on the growth of genetically matched high or low cyclin D1-expressing breast cancer cells was characterized and the interactions between cyclin D1, ER, and STAT3 in response to tamoxifen treatment were determined. We show that repression of STAT3 by cyclin D1 inhibits cell growth on Matrigel and in tumors in vivo; however, treatment with tamoxifen abolishes cyclin D1-mediated repression of STAT3 and growth suppression. We show that tamoxifen induces a redistribution of cyclin D1 from STAT3 to the ER, which results in the activation of both STAT3 and the ER. These results offer a molecular mechanism for the dual effect of cyclin D1 overexpression in breast cancer and support the notion that the level of cyclin D1 expression and activated STAT3 are important markers to predict response to tamoxifen treatment.