Stephanie A. Miller

β-catenin interacts with and inhibits NF-κB in human colon and breast cancer

Abstract β-catenin plays an important role in development and homeostasis. Deregulated β-catenin is involved in oncogenesis. In this study, we found that β-catenin can physically complex with NF-κB, resulting in a reduction of NF-κB DNA binding, transactivation activity, and target gene expression. Repressed NF-κB activity is found in human colon cancer cells in which β-catenin is activated. Importantly, activated β-catenin was found to inhibit the expression of NF-κB target genes, including Fas and TRAF1. Furthermore, a strong inverse correlation was identified between the expression levels of β-catenin and Fas in colon and breast tumor tissues, suggesting that β-catenin regulates NF-κB and its targets in vivo. Thus, β-catenin may play an important role in oncogenesis through the crossregulation of NF-κB.

The Crosstalk of mTOR/S6K1 and Hedgehog Pathways

Esophageal adenocarcinoma (EAC) is the most prevalent esophageal cancer type in the United States. The TNF-α/mTOR pathway is known to mediate the development of EAC. Additionally, aberrant activation of Gli1, downstream effector of the Hedgehog (HH) pathway, has been observed in EAC. In this study, we found that an activated mTOR/S6K1 pathway promotes Gli1 transcriptional activity and oncogenic function through S6K1-mediated Gli1 phosphorylation at Ser84, which releases Gli1 from its endogenous inhibitor, SuFu. Moreover, elimination of S6K1 activation by an mTOR pathway inhibitor enhances the killing effects of the HH pathway inhibitor. Together, our results established a crosstalk between the mTOR/S6K1 and HH pathways, which provides a mechanism for SMO-independent Gli1 activation and also a rationale for combination therapy for EAC.

Nuclear Expression of Epidermal Growth Factor Receptor is a Novel Prognostic Value in Patients With Ovarian Cancer

The epidermal growth factor receptor (EGFR) has previously been detected in the nucleus of cancer cells and primary tumors. We have reported that EGFR translocates from the plasma membrane to the nucleus. Accumulation of nuclear EGFR is linked to increased DNA synthesis and proliferation; however, the pathological significance of nuclear EGFR is not completely understood. Here, we sought to determine the predictive value of EGFR for the survival of ovarian cancer patients, through the examination of 221 cases of ovarian cancer tissues by immunohistochemical analysis to determine nuclear EGFR expression. In addition, we also examined cyclin D1 and Ki‐67 through immunohistochemisty. Furthermore, we examined nuclear EGFR levels in ovarian cancer cell lines treated with EGF, and primary ovarian tumor tissue using immunofluorescence analysis. Nuclear fractions extracted from serum‐starved cells treated with or without EGF were subjected to SDS–PAGE and Western blot analyses. We found that 28.3% of the cohort had high levels of nuclear EGFR, while 22.5% had low levels of nuclear EGFR, and 49.2% were negative for nuclear EGFR. Importantly, there was an inverse correlation between high nuclear EGFR, cyclin D1, and Ki‐67 with overall survival (P < 0.01, P < 0.09, P < 0.041). Additionally, nuclear EGFR correlated positively with increased levels of cyclin D1 and Ki‐67, both indicators for cell proliferation. Our findings indicate a pathological significance of nuclear EGFR that might be important for predicting clinical prognosis of ovarian cancer patients.

Mesenchymal Stem Cells Promote Formation of Colorectal Tumors in Mice

BACKGROUND & AIMS Tumor-initiating cells are a subset of tumor cells with the ability to form new tumors; however, they account for less than 0.001% of the cells in colorectal or other types of tumors. Mesenchymal stem cells (MSCs) integrate into the colorectal tumor stroma; we investigated their involvement in tumor initiation. METHODS Human colorectal cancer cells, MSCs, and a mixture of both cell types were injected subcutaneously into immunodeficient mice. We compared the ability of each injection to form tumors and investigated the signaling pathway involved in tumor initiation. RESULTS A small number (≤ 10) of unsorted, CD133⁻, CD166⁻, epithelial cell adhesion molecule⁻(EpCAM⁻), or CD133⁻/CD166⁻/EpCAM⁻ colorectal cancer cells, when mixed with otherwise nontumorigenic MSCs, formed tumors in mice. Secretion of interleukin (IL)-6 by MSCs increased the expression of CD133 and activation of Janus kinase 2-signal transducer and activator of transcription 3 (STAT3) in the cancer cells, and promoted sphere and tumor formation. An antibody against IL-6 or lentiviral-mediated transduction of an interfering RNA against IL-6 in MSCs or STAT3 in cancer cells prevented the ability of MSCs to promote sphere formation and tumor initiation. CONCLUSIONS IL-6, secreted by MSCs, signals through STAT3 to increase the numbers of colorectal tumor-initiating cells and promote tumor formation. Reagents developed to disrupt this process might be developed to treat patients with colorectal cancer.

Crossregulation of NF-κB by the APC/GSK-3β/β-catenin pathway

Glycogen synthase kinase‐3β (GSK‐3β) and adenomatous polyposis coli (APC) play an important role in the regulation of β‐catenin. Inhibition of or defects in their functions can lead to activation of β‐catenin. β‐catenin has been recently found to interact with and inhibit nuclear factor kappa B (NF‐κB). However, the regulatory roles of GSK‐3β/APC on the NF‐κB signaling pathway are unknown because of their diverse effects. In this study, we investigated whether GSK‐3β/APC might regulate NF‐κB activity through β‐catenin. We found that inhibition of GSK‐3β suppressed NF‐κB activity, whereas reexpression of APC restored NF‐κB activity in APC mutated cells. The regulatory effects were through β‐catenin because depletion of β‐catenin with small interfering RNA (siRNA) in the same systems reversed the effects. The regulatory relationship was further supported by the analysis of primary breast tumor tissues in vivo in which NF‐κB target TRAF1 was inversely correlated with activated β‐catenin. Thus, APC/GSK‐3β, through β‐catenin, may crossregulate NF‐κB signaling pathway.

Crossregulation of NF-kappaB by the APC/GSK-3beta/beta-catenin pathway.

Glycogen synthase kinase‐3β (GSK‐3β) and adenomatous polyposis coli (APC) play an important role in the regulation of β‐catenin. Inhibition of or defects in their functions can lead to activation of β‐catenin. β‐catenin has been recently found to interact with and inhibit nuclear factor kappa B (NF‐κB). However, the regulatory roles of GSK‐3β/APC on the NF‐κB signaling pathway are unknown because of their diverse effects. In this study, we investigated whether GSK‐3β/APC might regulate NF‐κB activity through β‐catenin. We found that inhibition of GSK‐3β suppressed NF‐κB activity, whereas reexpression of APC restored NF‐κB activity in APC mutated cells. The regulatory effects were through β‐catenin because depletion of β‐catenin with small interfering RNA (siRNA) in the same systems reversed the effects. The regulatory relationship was further supported by the analysis of primary breast tumor tissues in vivo in which NF‐κB target TRAF1 was inversely correlated with activated β‐catenin. Thus, APC/GSK‐3β, through β‐catenin, may crossregulate NF‐κB signaling pathway.

Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

Receptor tyrosine kinases (RTKs) are cell surface receptors that initiate signal cascades in response to ligand stimulation. Abnormal expression and dysregulated intracellular trafficking of RTKs have been shown to be involved in tumorigenesis. Recent evidence shows that these cell surface receptors translocate from cell surface to different cellular compartments, including the Golgi, mitochondria, endoplasmic reticulum (ER) and the nucleus, to regulate physiological and pathological functions. Although some trafficking mechanisms have been resolved, the mechanism of intracellular trafficking from cell surface to the Golgi is not yet completely understood. Here we report a mechanism of Golgi translocation of epidermal growth factor receptor (EGFR) in which EGF-induced EGFR travels to the Golgi via microtubule-dependent movement by interacting with dynein and fuses with the Golgi through syntaxin 6-mediated membrane fusion. We also demonstrate that the microtubule- and syntaxin 6-mediated Golgi translocation of EGFR is necessary for its consequent nuclear translocation and nuclear functions. Thus, together with previous studies, the microtubule- and syntaxin 6-mediated trafficking pathway from cell surface to the Golgi, ER and the nucleus defines a comprehensive trafficking route for EGFR to travel from cell surface to the Golgi and the nucleus.

Colon cancer stem cells resist antiangiogenesis therapy-induced apoptosis

Antiangiogenesis is an efficient therapy for eliminating colon cancers, but because of recurrence it remains only palliative. We hypothesized that certain populations of tumor cells resist antiangiogenesis-induced apoptosis and explored the underlying mechanism. We demonstrated that the CD133(+) population of cells in colon cancer is resistant to anti-angiogenesis therapy. Additionally, we identified an anti-apoptotic signaling pathway responsible for this resistance involving PP2A, p38MAPK, MAPKAPK2, and Hsp27. Thus, this pathway may offer a new avenue to develop target therapy for colorectal cancer.

Survival of Cancer Stem Cells under Hypoxia and Serum Depletion via Decrease in PP2A Activity and Activation of p38-MAPKAPK2-Hsp27

Hypoxia and serum depletion are common features of solid tumors that occur upon antiangiogenesis, irradiation and chemotherapy across a wide variety of malignancies. Here we show that tumor cells expressing CD133, a marker for colorectal cancer initiating or stem cells, are enriched and survive under hypoxia and serum depletion conditions, whereas CD133− cells undergo apoptosis. CD133+ tumor cells increase cancer stem cell and epithelial-mesenchymal transition properties. Moreover, via screening a panel of tyrosine and serine/threonine kinase pathways, we identified Hsp27 is constitutively activated in CD133+ cells rather than CD133− cell under hypoxia and serum depletion conditions. However, there was no difference in Hsp27 activation between CD133+ and CD133− cells under normal growth condition. Hsp27 activation, which was mediated by the p38MAPK-MAPKAPK2-Hsp27 pathway, is required for CD133+ cells to inhibit caspase 9 and 3 cleavage. In addition, inhibition of Hsp27 signaling sensitizes CD133+ cells to hypoxia and serum depletion -induced apoptosis. Moreover, the antiapoptotic pathway is also activated in spheroid culture-enriched CD133+ cancer stem cells from a variety of solid tumor cells including lung, brain and oral cancer, suggesting it is a common pathway activated in cancer stem cells from multiple tumor types. Thus, activation of PP2A or inactivation of the p38MAPK-MAPKAPK2-Hsp27 pathway may develop new strategies for cancer therapy by suppression of their TIC population.

Crossregulation of NF-?B by the APC/GSK-3?/?-catenin pathway

Glycogen synthase kinase‐3β (GSK‐3β) and adenomatous polyposis coli (APC) play an important role in the regulation of β‐catenin. Inhibition of or defects in their functions can lead to activation of β‐catenin. β‐catenin has been recently found to interact with and inhibit nuclear factor kappa B (NF‐κB). However, the regulatory roles of GSK‐3β/APC on the NF‐κB signaling pathway are unknown because of their diverse effects. In this study, we investigated whether GSK‐3β/APC might regulate NF‐κB activity through β‐catenin. We found that inhibition of GSK‐3β suppressed NF‐κB activity, whereas reexpression of APC restored NF‐κB activity in APC mutated cells. The regulatory effects were through β‐catenin because depletion of β‐catenin with small interfering RNA (siRNA) in the same systems reversed the effects. The regulatory relationship was further supported by the analysis of primary breast tumor tissues in vivo in which NF‐κB target TRAF1 was inversely correlated with activated β‐catenin. Thus, APC/GSK‐3β, through β‐catenin, may crossregulate NF‐κB signaling pathway.