Versican is highly expressed during the early stages of tissue development and its expression is elevated during wound repair and tumor growth. There is little literature on the potential role of breast cancer stem cells on the cellular–extracellular matrix interactions involving versican. An anti-versican short hairpin RNA (shRNA) was used to observe the effect of reduction of versican on breast cancer self-renewal. A versican G3 construct was exogenously expressed in breast cancer cell lines. Colony formation and mammosphere formation assays were conducted; flow cytometry was applied to analyze the prevalence of side population cells. The versican G3- and vector-transfected 66c14 cells were injected transdermally into BALB/c mice as a 10-fold dilution series from 1 x 105 to 1 x 102 cells per mouse. Versican G3 domain enhanced breast cancer self-renewal in both experimental in vitro and in vivo models. Versican G3–transfected cells contained high levels of side population cells, formed more mammospheres when cultured in the serum-free medium, and formed a greater number and larger colonies. Reduction of versican's functionality through anti-versican shRNA or knocking out the EGF-like motifs reduced the effect of versican on enhancing mammosphere and colony formation. Versican-enhanced self-renewal played a role in enhanced chemotherapeutic drug resistance, relating partly to the upregulated expression of EGF receptor (EGFR) signaling. Versican is highly expressed in breast cancer progenitor cells and was maintained at high levels before cell differentiation. Overexpression of versican enhanced breast cancer self-renewal through EGFR/AKT/GSK-3β (S9P) signaling and conferred resistant to chemotherapeutic drugs tested. Mol Cancer Res; 11(5); 443–55. ©2013 AACR.
Metastasis is the leading cause of death among patients who have breast cancer. Understanding the role of the extracellular matrix (ECM) in the metastatic process may lead to the development of improved therapies to treat patients with cancer. Intratumoral hypoxia, found in the majority of breast cancers, is associated with an increased risk of metastasis and mortality. We found that in hypoxic breast cancer cells, hypoxia-inducible factor 1 (HIF-1) activates transcription of the PLOD1 and PLOD2 genes encoding procollagen lysyl hydroxylases that are required for the biogenesis of collagen, which is a major constituent of the ECM. High PLOD2 expression in breast cancer biopsies is associated with increased risk of mortality. We show that PLOD2 is critical for fibrillar collagen formation by breast cancer cells, increases tumor stiffness, and is required for metastasis to lymph nodes and lungs. Mol Cancer Res; 11(5); 456–66. ©2013 AACR.
Tuberous sclerosis complex (TSC) is a hamartoma syndrome in which brain, renal, and lung tumors develop and cause both morbidity and death. Loss of either TSC1 or TSC2 in TSC hamartomas leads to activation of mTORC1. Rapamycin and related drugs have been shown to have clinical benefit for these tumors in patients with TSC and those with sporadic forms of TSC-related neoplasms. However, lifelong therapy seems to be required, as tumors are not eliminated by this treatment. We examined the potential benefit of MLN0128, a novel potent mTOR ATP-competitive inhibitor, as a therapeutic strategy for renal cystadenomas that develop in A/J Tsc2+/– mice. Rapamycin given by intraperitoneal injection at 3 mg/kg 3 times per week, and MLN0128 given by gavage at 0.75 mg/kg 5 times per week had equivalent effects in suppressing tumor development during a 4-week treatment period, with an approximate 99% reduction in microscopic tumor cell volume. Marked reduction in activation of mTOR complex (mTORC)1 and blockade of cell growth was seen with both drugs, whereas only MLN0128 treatment had effects in blocking mTORC2 and 4EBP1 phosphorylation. However, when either drug was discontinued and mice were observed for two additional months, there was dramatic recovery of tumor growth, with extensive proliferation. Hence, longlasting tumor growth control is not achieved with transient treatment with either drug, and MLN0128 and rapamycin have equivalent therapeutic benefit in this mouse model. Differences in side-effect profiles might make MLN0128 more attractive for treatment of patients with TSC-related tumors, but will require additional study in humans. Mol Cancer Res; 11(5); 467–73. ©2013 AACR.
mTOR has been implicated in survival signals for many human cancers. Rapamycin and TGF-β synergistically induce G1 cell-cycle arrest in several cell lines with intact TGF-β signaling pathway, which protects cells from the apoptotic effects of rapamycin during S-phase of the cell cycle. Thus, rapamycin is cytostatic in the presence of serum/TGF-β and cytotoxic in the absence of serum. However, if TGF-β signaling is defective, rapamycin induced apoptosis in both the presence and absence of serum/TGF-β in colon and breast cancer cell lines. Because genetic dysregulation of TGF-β signaling is commonly observed in pancreatic cancers—with defects in the Smad4 gene being most prevalent, we hypothesized that pancreatic cancers would display a synthetic lethality to rapamycin in the presence of serum/TGF-β. We report here that Smad4-deficient pancreatic cancer cells are killed by rapamycin in the absence of serum; however, in the presence of serum, we did not observe the predicted synthetic lethality with rapamycin. Rapamycin also induced elevated phosphorylation of the survival kinase Akt at Ser473. Suppression of rapamycin-induced Akt phosphorylation restored rapamycin sensitivity in Smad4-null, but not Smad4 wild-type pancreatic cancer cells. This study shows that the synthetic lethality to rapamycin in pancreatic cancers with defective TGF-β signaling is masked by rapamycin-induced increases in Akt phosphorylation. The implication is that a combination of approaches that suppress both Akt phosphorylation and mTOR could be effective in targeting pancreatic cancers with defective TGF-β signaling. Mol Cancer Res; 11(5); 474–81. ©2013 AACR.
Advanced prostate cancer is characterized by incurable castration-resistant progression and osteoblastic bone metastasis. While androgen deprivation therapy remains the primary treatment for advanced prostate cancer, resistance inevitably develops. Importantly, mounting evidence indicates that androgen receptor (AR) signaling continues to play a critical role in the growth of advanced prostate cancer despite androgen deprivation. While the mechanisms of aberrant AR activation in advanced prostate cancer have been extensively studied, the downstream AR target genes involved in the progression of castration resistance are largely unknown. Here, we identify WNT7B as a direct AR target gene highly expressed in castration-resistant prostate cancer (CRPC) cells. Our results show that expression of WNT7B is necessary for the growth of prostate cancer cells and that this effect is enhanced under androgen-deprived conditions. Further analyses reveal that WNT7B promotes androgen-independent growth of CRPC cells likely through the activation of protein kinase C isozymes. Our results also show that prostate cancer-produced WNT7B induces osteoblast differentiation in vitro through a direct cell–cell interaction, and that WNT7B is upregulated in human prostate cancer xenografts that cause an osteoblastic reaction when grown in bone. Taken together, these results suggest that AR-regulated WNT7B signaling is critical for the growth of CRPC and development of the osteoblastic bone response characteristic of advanced prostate cancer. Mol Cancer Res; 11(5); 482–93. ©2013 AACR.
Glioblastoma tumors are characterized by their invasiveness and resistance to therapies. The transcription factor signal transducer and activator of transcription 3 (STAT3) was recently identified as a master transcriptional regulator in the mesenchymal subtype of glioblastoma (GBM), which has generated an increased interest in targeting STAT3. We have evaluated more closely the mechanism of action of one particular STAT3 inhibitor, JSI-124 (cucurbitacin I). In this study, we confirmed that JSI-124 inhibits both constitutive and stimulus-induced Janus kinase 2 (JAK2) and STAT3 phosphorylation, and decreases cell proliferation while inducing apoptosis in cultured GBM cells. However, we discovered that before the inhibition of STAT3, JSI-124 activates the nuclear factor-B (NF-B) pathway, via NF-B p65 phosphorylation and nuclear translocation. In addition, JSI-124 treatment induces the expression of IL-6, IL-8, and suppressor of cytokine signaling (SOCS3) mRNA, which leads to a corresponding increase in IL-6, IL-8, and SOCS3 protein expression. Moreover, the NF-B–driven SOCS3 expression acts as a negative regulator of STAT3, abrogating any subsequent STAT3 activation and provides a mechanism of STAT3 inhibition after JSI-124 treatment. Chromatin immunoprecipitation analysis confirms that NF-B p65 in addition to other activating cofactors are found at the promoters of IL-6, IL-8, and SOCS3 after JSI-124 treatment. Using pharmacological inhibition of NF-B and inducible knockdown of NF-B p65, we found that JSI-124–induced expression of IL-6, IL-8, and SOCS3 was significantly inhibited, showing an NF-B–dependent mechanism. Our data indicate that although JSI-124 may show potential antitumor effects through inhibition of STAT3, other off-target proinflammatory pathways are activated, emphasizing that more careful and thorough preclinical investigations must be implemented to prevent potential harmful effects. Mol Cancer Res; 11(5); 494–505. ©2013 AACR.
Heparin-binding EGF-like growth factor (HB-EGF) is one of several proangiogenic factors and represents a possible therapeutic target for patients with triple-negative breast cancer (TNBC). However, the role of HB-EGF in promoting tumor aggressiveness in TNBC remains unclear. To investigate specific genes and pathways involved in TNBC tumorigenesis, we profiled gene expression changes in two TNBC cell lines under two-dimensional culture (2DC) and three-dimensional culture (3DC) and in a tumor xenograft model. We identified simultaneous upregulation of HB-EGF, VEGFA, and angiopoietin-like 4 (ANGPTL4) in 3DC and tumor xenografts, compared with 2DC. We show that HB-EGF regulates the expression of VEGFA or ANGPTL4 via transcriptional regulation of hypoxia-inducible factor-1α and NF-B. Furthermore, suppression of VEGFA or ANGPTL4 expression enhanced HB-EGF expression, highlighting a unique regulatory loop underlying this angiogenesis network. Targeted knockdown of HB-EGF significantly suppressed tumor formation in a TNBC xenograft model, compared with individual knockdown of either VEGFA or ANGPTL4, by reducing the expression of both VEGFA and ANGPTL4. In patients with TNBC, VEGFA or ANGPTL4 expression was also significantly correlated with HB-EGF expression. Low concentrations of exogenously added HB-EGF strongly activated the proliferation of endothelial cells, tube formation, and vascular permeability in blood vessels, in a similar fashion to high doses of VEGFA and ANGPTL4. Taken together, these results suggest that HB-EGF plays a pivotal role in the acquisition of tumor aggressiveness in TNBC by orchestrating a molecular hierarchy regulating tumor angiogenesis. Mol Cancer Res; 11(5); 506–17. ©2013 AACR.
Glutathione S-transferase mu2 (GST-M2) is a phase II detoxification enzyme. Low expression of GST-M2 in lung cancers is due to hypermethylation of its promoter. Lung cancer with the GST mu-null genotype is associated with shorter survival. However, a correlation between GST-M2 and important clinical parameters, as well as the migration of GST-M2–defective cells in lung cancer, has not been established. In the present study, we investigate the role of GST-M2 in cell migration and actin disassembly in lung cancer cells. GST-M2 and CCN2 mRNA levels were significantly reduced in non–small cell lung cancer (NSCLC) tumors when compared with matched normal lung tissues in 82 patients with NSCLC. We found that high expressions of both GST-M2 and CCN2 are correlated with favorable survival of patients with lung cancer when compared with similar patients without GST-M2 or CCN2 expression. GST-M2 can induce CCN2 expression by driving the CCN2 proximal promoter. Overexpression of GST-M2 decreases the formation of filopodia, resulting in remodeling of the reorganized cytoskeletons. Overexpression of GST-M2 significantly suppressed cancer cell migration on wound-healing assay. In addition, overexpression of GST-M2 dramatically reduced tumor growth and metastasis in a xenograft mouse model. These data highlight the potential of GST-M2 as a novel tumor suppressor. GST-M2 increases the expression of CCN2 in lung cancer cells, which inhibits cancer cell migration in lung cancer and animal models. Mol Cancer Res; 11(5); 518–29. ©2013 AACR.
During metastatic progression, an aberrant epithelial-to-mesenchymal transformation (EMT) that is most often driven by the loss of the cell–cell adhesion molecule E-cadherin generates noncohesive tumor cells that are highly invasive. We used mesenchymally transformed, E-cadherin–negative MDA-MB-231 breast carcinoma cells in a natural product screen and determined that the triterpenoid saponin sarasinoside A1 inhibited their invasion and the invasion of a number of other tumor cell lines. Sarasinoside A1 also caused MDA-MB-231 cells to become cohesive in a three-dimensional basement membrane and collagen gel cultures. In two-dimensional culture, sarasinoside A1 initiated a morphologic re-epithelialization of MDA-MB-231 cells wherein preexisting nonepithelial cadherins and the junction-associated proteins β-catenin and ZO-1 all relocalized to sites of cell–cell contact. In addition, the intercellular space between neighboring cells narrowed considerably, the stability of polymerized actin at cell-cell contact sites increased, and there was a recruitment and stabilization of nectin-based adhesion complexes to these sites, all of which strongly suggested that functional cell–cell junctions had formed. Importantly, sarasinoside A1 induced nascent cell–cell junction formation that did not require changes in gene expression and was not associated with an induction of E-cadherin but resulted in increased activation of Rap GTPases. Therefore, our findings with sarasinoside A1 suggest that it may be possible to re-epithelialize metastatic tumor cells with phenotypic consequence even when E-cadherin is completely absent. Mol Cancer Res; 11(5); 530–40. ©2013 AACR.
B-cell receptor (BCR) signals promote survival of chronic lymphocytic leukemia (CLL) cells, and it is believed that overexpressed and constitutively active Lyn mediates this signaling. Here, we show that CLL cells express lymphocyte-specific protein tyrosine kinase (LCK) and that inhibition of this Src family tyrosine kinase with the specific inhibitor [4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[3,2-d]pyrimidin-7-yl-cyclopentane (Lck-i)], or reduction of its expression with siRNA, blocks the induction of CD79a, Syk, inhibitor of IB kinase (IKK), Akt, and extracellular signal–regulated kinase (ERK) phosphorylation by BCR cross-linking in these cells. Furthermore, we show that CLL cells with high levels of LCK expression have higher levels of BCR-mediated IKK, Akt, and ERK phosphorylation as well as cell survival than CLL cells with low levels of LCK expression. We also show that treatment of CLL cells with Lck-i inhibits BCR cross-linking–induced cell survival. Taken together, these data show a major role for LCK in proximal and distal BCR-mediated signaling in CLL cells and suggest that LCK expression is important in the pathogenesis of this disease. On a clinical level, these studies advocate the use of specific LCK inhibitors in the treatment of progressive CLL. Mol Cancer Res; 11(5); 541–54. ©2013 AACR.
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