Michele I. Vitolo

Knockin of mutant PIK3CA activates multiple oncogenic pathways

The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. Here we used gene targeting to “knock in” PIK3CA mutations into human breast epithelial cells to identify new therapeutic targets associated with oncogenic PIK3CA. Mutant PIK3CA knockin cells were capable of epidermal growth factor and mTOR-independent cell proliferation that was associated with AKT, ERK, and GSK3β phosphorylation. Paradoxically, the GSK3β inhibitors lithium chloride and SB216763 selectively decreased the proliferation of human breast and colorectal cancer cell lines with oncogenic PIK3CA mutations and led to a decrease in the GSK3β target gene CYCLIN D1. Oral treatment with lithium preferentially inhibited the growth of nude mouse xenografts of HCT-116 colon cancer cells with mutant PIK3CA compared with isogenic HCT-116 knockout cells containing only wild-type PIK3CA. Our findings suggest GSK3β is an important effector of mutant PIK3CA, and that lithium, an FDA-approved therapy for bipolar disorders, has selective antineoplastic properties against cancers that harbor these mutations.

Tamoxifen-stimulated growth of breast cancer due to p21 loss

Tamoxifen is widely used for the treatment of hormonally responsive breast cancers. However, some resistant breast cancers develop a growth proliferative response to this drug, as evidenced by tumor regression upon its withdrawal. To elucidate the molecular mediators of this paradox, tissue samples from a patient with tamoxifen-stimulated breast cancer were analyzed. These studies revealed that loss of the cyclin-dependent kinase inhibitor p21 was associated with a tamoxifen growth-inducing phenotype. Immortalized human breast epithelial cells with somatic deletion of the p21 gene were then generated and displayed a growth proliferative response to tamoxifen, whereas p21 wild-type cells demonstrated growth inhibition upon tamoxifen exposure. Mutational and biochemical analyses revealed that loss of p21s cyclin-dependent kinase inhibitory property results in hyperphosphorylation of estrogen receptor-α, with subsequent increased gene expression of estrogen receptor-regulated genes. These data reveal a previously uncharacterized molecular mechanism of tamoxifen resistance and have potential clinical implications for the management of tamoxifen-resistant breast cancers.

Beta-platelet-derived growth factor receptor mediates motility and growth of Ewing's sarcoma cells

The Ewings sarcoma family of tumors (ESFT) contain a translocation, t(11;22), which results in the novel oncogenic fusion protein EWS/FLI1. Platelet-derived growth factors (PDGF) and their receptors (PDGFR) are involved in the induction and proliferation of numerous solid tumors and are the potential candidates for novel targeted antitumor therapy. Since a relation was reported between PDGF-C and EWS/FLI1, we sought to characterize the PDGF signaling pathway in ESFT. Eight out of nine ESFT cell lines were found to express significant levels of β-PDGFR. Interestingly, none of the tested cell lines expressed α-PDGFR, which is the receptor isotype required for PDGF-C binding. By immunohistochemical staining 47 of 52 (90.4%) archival tumor samples from patients with ESFT were positive for β-PDGFR. ESFT cell lines were treated with PDGF-AA or PDGF-BB ligands to evaluate downstream signaling. Autophosphorylation of β-PDGFR and tyrosine phosphorylation of PLC-γ, PI3Kp85 and Shc were detected only in PDGF-BB-stimulated cells that express β-PDGFR. Receptor function was further evaluated using chemotaxis assays that showed TC-32 cell migration towards PDGF-BB. A specific PDGFR kinase inhibitor AG1295 blocked β-PDGFR activation, downstream signaling, growth in cell culture and chemotaxis of TC-32 cells. AG1295 also delayed tumor formation and prolonged survival in an ESFT animal model. We conclude that ESFT express β-PDGFR and that this is a functional and potentially crucial signaling pathway. Therefore, β-PDGFRs may provide a novel therapeutic target in ESFT that can be utilized to design better treatment modalities.

Deletion of PTEN Promotes Tumorigenic Signaling, Resistance to Anoikis, and Altered Response to Chemotherapeutic Agents in Human Mammary Epithelial Cells

Many cancers, including breast cancer, harbor loss-of-function mutations in the catalytic domain of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or have reduced PTEN expression through loss of heterozygosity and/or epigenetic silencing mechanisms. However, specific phenotypic effects of PTEN inactivation in human cancer cells remain poorly defined without a direct causal connection between the loss of PTEN function and the development or progression of cancer. To evaluate the biological and clinical relevance of reduced or deleted PTEN expression, a novel in vitro model system was generated using human somatic cell knockout technologies. Targeted homologous recombination allowed for a single and double allelic deletion, which resulted in reduced and deleted PTEN expression, respectively. We determined that heterozygous loss of PTEN in the nontumorigenic human mammary epithelial cell line MCF-10A was sufficient for activation of the phosphoinositide 3-kinase/AKT and mitogen-activated protein kinase pathways, whereas the homozygous absence of PTEN expression led to a further increased activation of both pathways. The deletion of PTEN was able to confer growth factor-independent proliferation, which was confirmed by the resistance of the PTEN(-/-) MCF-10A cells to small-molecule inhibitors of the epidermal growth factor receptor. However, neither heterozygous nor homozygous loss of PTEN expression was sufficient to promote anchorage-independent growth, but the loss of PTEN did confer apoptotic resistance to cell rounding and matrix detachment. Finally, MCF-10A cells with the reduction or loss of PTEN showed increased susceptibility to the chemotherapeutic drug doxorubicin but not paclitaxel.

Metastatic breast tumors express increased tau, which promotes microtentacle formation and the reattachment of detached breast tumor cells.

The cytoskeletal organization of detached and circulating tumor cells (CTCs) is currently not well defined and may provide potential targets for new therapies to limit metastatic tumor spread. In vivo, CTCs reattach in distant tissues by a mechanism that is tubulin-dependent and suppressed by polymerized actin. The cytoskeletal mechanisms that promote reattachment of CTCs match exactly with the mechanisms supporting tubulin microtentacles (McTN), which we have recently identified in detached breast tumor cells. In this study, we aimed to investigate how McTN formation is affected by the microtubule-associated protein, tau, which is expressed in a subset of chemotherapy-resistant breast cancers. We demonstrate that endogenous tau protein localizes to McTNs and is both necessary and sufficient to promote McTN extension in detached breast tumor cells. Tau-induced McTNs increase reattachment of suspended cells and retention of CTCs in lung capillaries. Analysis of patient-matched primary and metastatic tumors reveals that 52% possess tau expression in metastases and 26% display significantly increased tau expression over disease progression. Tau enrichment in metastatic tumors and the ability of tau to promote tumor cell reattachment through McTN formation support a model in which tau-induced microtubule stabilization provides a selective advantage during tumor metastasis.

Curcumin Targets Breast Cancer Stem–like Cells with Microtentacles That Persist in Mammospheres and Promote Reattachment

Cancer stem-like cells (CSC) and circulating tumor cells (CTC) have related properties associated with distant metastasis, but the mechanisms through which CSCs promote metastasis are unclear. In this study, we report that breast cancer cell lines with more stem-like properties display higher levels of microtentacles (McTN), a type of tubulin-based protrusion of the plasma cell membrane that forms on detached or suspended cells and aid in cell reattachment. We hypothesized that CSCs with large numbers of McTNs would more efficiently attach to distant tissues, promoting metastatic efficiency. The naturally occurring stem-like subpopulation of the human mammary epithelial (HMLE) cell line presents increased McTNs compared with its isogenic non-stem-like subpopulation. This increase was supported by elevated α-tubulin detyrosination and vimentin protein levels and organization. Increased McTNs in stem-like HMLEs promoted a faster initial reattachment of suspended cells that was inhibited by the tubulin-directed drug, colchicine, confirming a functional role for McTNs in stem cell reattachment. Moreover, live-cell confocal microscopy showed that McTNs persist in breast stem cell mammospheres as flexible, motile protrusions on the surface of the mammosphere. Although exposed to the environment, they also function as extensions between adjacent cells along cell-cell junctions. We found that treatment with the breast CSC-targeting compound curcumin rapidly extinguished McTN in breast CSC, preventing reattachment from suspension. Together, our results support a model in which breast CSCs with cytoskeletal alterations that promote McTNs can mediate attachment and metastasis but might be targeted by curcumin as an antimetastatic strategy.

Regulation of TGFβ1-mediated growth inhibition and apoptosis by RUNX2 isoforms in endothelial cells

Runx transcription factors regulate viral growth, hematopoiesis, bone formation, angiogenesis, and gastric epithelial development through specific DNA-binding motifs on target gene promoters. Vascular endothelial cells (ECs) express RUNX genes that are activated by angiogenic factors. The RUNX2 gene also activates the vascular endothelial growth factor promoter. Alternatively spliced forms of RUNX genes have been described, but their functions in angiogenesis have not been elucidated. In this study, expression of a novel alternatively spliced variant of RUNX2 (RUNX2Δ8), lacking the region encoded by exon 8, was detected in aortic tissue undergoing angiogenesis in vitro and in ECs. Expression of RUNX2 and RUNX2Δ8 increased in vascular sprouts concomitant with expression of cellular proteases and cytokines known to mediate angiogenesis. RUNX2 DNA-binding activity was expressed in proliferating but not quiescent ECs. Ectopic expression of RUNX2 in ECs increased cell sprouting, cell proliferation, DNA synthesis, and phosphorylation of phosphorylated retinoblastoma relative to control transfectants while RUNX2, but not RUNX2Δ8 transfectants, acquired resistance to growth inhibition by transforming growth factor (TGFβ1). Furthermore, RUNX2Δ8-transfected cells were more sensitive to TGFβ1-induced apoptosis than RUNX2 transfectants. Consistent with these data, the RUNX2 gene was a strong repressor of the promoter of the cyclin-dependent kinase inhibitor, p21CIP1, while RUNX2Δ8 was a competitive inhibitor of RUNX2 and exhibited weak repression activity. These results support the hypothesis that ECs regulate growth and apoptosis, in part, by alternative splicing events in the RUNX2 transcription factor to affect the TGFβ1 signaling pathway. The exon 8 domain of RUNX2 may contribute to the strong repression activity of RUNX2 for some target gene promoters.

The RUNX2 transcription factor cooperates with the YES-associated protein, YAP65, to promote cell transformation

The Runt box domain DNA‑binding transcription factors (RUNX) play key roles in hematopoietic, bone, and gastric development. These factors regulate angiogenesis and tumorigenic events, functioning as either activators or repressors of target genes. Although RUNX2 is an essential bone maturation factor, it has also been found to promote transformation in vivo and cell proliferation in vitro, perhaps by associating with specific coactivators or corepressors. Adenoviral‑mediated overexpression of dominant negative RUNX2 or specific reduction of RUNX2 with RNA‑interference inhibits cell proliferation. To determine whether RUNX2 also plays a role in cell transformation, RUNX2 interactions with the coactivator Yes‑associated protein (YAP65) were examined. RUNX2 associated with YAP65 via a proline‑rich segment in the C‑terminal domain (PPPY) and coexpression of RUNX2 and YAP65 significantly increased foci formation and anchorage‑independent growth relative to each factor alone. However, in contrast to wild‑type RUNX2, a mutant RUNX2(P409A), which does not bind YAP65, did not cooperate with YAP65 to promote anchorage‑independent growth. RUNX2 is a strong repressor of the cyclin‑dependent kinase inhibitor p21CIP1, which is known to mediate cell transformation. Overexpression of YAP65 prevented RUNX2‑dependent downregulation of p21CIP1 protein expression while promoting cell transformation. The RUNX2(P409A) mutant retained the ability to bind DNA and repress the p21CIP1 promoter as shown by DNA precipitation and luciferase‑reporter assays, respectively, but was not able to relieve repression of the p21CIP1 promoter. Therefore, these results reveal a novel function of the RUNX2 and YAP65 interaction in oncogenic transformation that may be mediated by modulation of p21CIP1 protein expression.

α-Tubulin Acetylation Elevated in Metastatic and Basal-like Breast Cancer Cells Promotes Microtentacle Formation, Adhesion, and Invasive Migration

Metastatic cases of breast cancer pose the primary challenge in clinical management of this disease, demanding the identification of effective therapeutic strategies that remain wanting. In this study, we report that elevated levels of α-tubulin acetylation are a sufficient cause of metastatic potential in breast cancer. In suspended cell culture conditions, metastatic breast cancer cells exhibited high α-tubulin acetylation levels that extended along microtentacle (McTN) protrusions. Mutation of the acetylation site on α-tubulin and enzymatic modulation of this posttranslational modification exerted a significant impact on McTN frequency and the reattachment of suspended tumor cells. Reducing α-tubulin acetylation significantly inhibited migration but did not affect proliferation. In an analysis of more than 140 matched primary and metastatic tumors from patients, we found that acetylation was maintained and in many cases increased in lymph node metastases compared with primary tumors. Proteomic analysis of an independent cohort of more than 390 patient specimens further documented the relationship between increased α-tubulin acetylation and the aggressive behaviors of basal-like breast cancers, with a trend toward increased risk of disease progression and death in patients with high-intensity α-tubulin acetylation in primary tumors. Taken together, our results identify a tight correlation between acetylated α-tubulin levels and aggressive metastatic behavior in breast cancer, with potential implications for the definition of a simple prognostic biomarker in patients with breast cancer.

MCT-1 Protein Interacts with the Cap Complex and Modulates Messenger RNA Translational Profiles

MCT-1 is an oncogene that was initially identified in a human T cell lymphoma and has been shown to induce cell proliferation as well as activate survival-related pathways. MCT-1 contains the PUA domain, a recently described RNA-binding domain that is found in several tRNA and rRNA modification enzymes. Here, we established that MCT-1 protein interacts with the cap complex through its PUA domain and recruits the density-regulated protein (DENR/DRP), containing the SUI1 translation initiation domain. Through the use of microarray analysis on polysome-associated mRNAs, we showed that up-regulation of MCT-1 was able to modulate the translation profiles of BCL2L2, TFDP1, MRE11A, cyclin D1, and E2F1 mRNAs, despite equivalent levels of mRNAs in the cytoplasm. Our data establish a role for MCT-1 in translational regulation, and support a linkage between translational control and oncogenesis.