Theresa Nguyen

Vimentin is a novel AKT1 target mediating motility and invasion

The PI3K/AKT signaling pathway is aberrant in a wide variety of cancers. Downstream effectors of AKT are involved in survival, growth and metabolic-related pathways. In contrast, contradictory data relating to AKT effects on cell motility and invasion, crucial prometastatic processes, have been reported pointing to a potential cell type and isoform type-specific AKT-driven function. By implication, study of AKT signaling should optimally be conducted in an appropriate intracellular environment. Prognosis in soft-tissue sarcoma (STS), the aggressive malignancies of mesenchymal origin, is poor, reflecting our modest ability to control metastasis, an effort hampered by lack of insight into molecular mechanisms driving STS progression and dissemination. We examined the impact of the cancer progression-relevant AKT pathway on the mesenchymal tumor cell internal milieu. We demonstrate that AKT1 activation induces STS cell motility and invasiveness at least partially through a novel interaction with the intermediate filament vimentin (Vim). The binding of AKT (tail region) to Vim (head region) results in Vim Ser39 phosphorylation enhancing the ability of Vim to induce motility and invasion while protecting Vim from caspase-induced proteolysis. Moreover, vimentin phosphorylation was shown to enhance tumor and metastasis growth in vivo. Insights into this mesenchymal-related molecular mechanism may facilitate the development of critically lacking therapeutic options for these devastating malignancies.

Vascular Endothelial Growth Factor Overexpression by Soft Tissue Sarcoma Cells: Implications for Tumor Growth, Metastasis, and Chemoresistance

To better elucidate the role of vascular endothelial growth factor (VEGF)(165) in soft tissue sarcoma (STS) growth, metastasis, and chemoresistance, we generated stably transfected human STS cell lines with VEGF(165) to study the effect of VEGF(165) on STS cells in vitro and the effect of culture medium from these cells on human umbilical vascular endothelial cells. Severe combined immunodeficient mice bearing xenografts of transfected cell lines were used to assess the effect of VEGF overexpression and the effect of VEGF receptor (VEGFR) 2 inhibition on STS growth, metastasis, and response to doxorubicin. VEGF(165)-transfected xenografts formed highly vascular tumors with shorter latency, accelerated growth, enhanced chemoresistance, and increased incidence of pulmonary metastases. Blockade of VEGFR2 signaling using DC101 anti-VEGFR2 monoclonal antibody enhanced doxorubicin chemoresponse; this combined biochemotherapy inhibited tumor growth and decreased pulmonary metastases without overt toxicity. Combined therapy reduced microvessel counts while increasing vessel maturation index. VEGF overexpression did not affect on the sarcoma cells per se; however, conditioned medium from VEGF transfectants caused increased endothelial cell proliferation, migration, and chemoresistance. Addition of DC101 induced endothelial cell sensitivity to doxorubicin and suppressed the activity of matrix metalloproteinases secreted by endothelial cells. We therefore conclude that VEGF is a critical determinant of STS growth and metastasis and that STS chemoresistance, in our model, is a process induced by the interplay between STS cells and tumor-associated endothelial cells. STS growth and metastasis can be interrupted by combined low-dose doxorubicin and anti-VEGFR2, a strategy that attacks STS-associated endothelial cells. In the future, such therapeutic approaches may be useful in treating STS before the development of clinically apparent metastases.

Activated MET is a molecular prognosticator and potential therapeutic target for malignant peripheral nerve sheath tumors

Purpose: MET signaling has been suggested a potential role in malignant peripheral nerve sheath tumors (MPNST). Here, MET function and blockade were preclinically assessed. Experimental Design: Expression levels of MET, its ligand hepatocyte growth factor (HGF), and phosphorylated MET (pMET) were examined in a clinically annotated MPNST tissue microarray (TMA) incorporating univariable and multivariable statistical analyses. Human MPNST cells were studied in vitro and in vivo; Western blot (WB) and ELISA were used to evaluate MET and HGF expression, activation, and downstream signaling. Cell culture assays tested the impact of HGF-induced MET activation and anti-MET–specific siRNA inhibition on cell proliferation, migration, and invasion; in vivo gel-foam assays were used to evaluate angiogenesis. Cells stably transduced with anti-MET short hairpin RNA (shRNA) constructs were tested for growth and metastasis in severe combined immunodeficient (SCID) mice. The effect of the tyrosine kinase inhibitor XL184 (Exelixis) targeting MET/VEGFR2 (vascular endothelial growth factor receptor 2) on local and metastatic MPNST growth was examined in vivo. Results: All three markers were expressed in MPNST human samples; pMET expression was an independent prognosticator of poor patient outcome. Human MPNST cell lines expressed MET, HGF, and pMET. MET activation increased MPNST cell motility, invasion, angiogenesis, and induced matrix metalloproteinase-2 (MMP2) and VEGF expression; MET knockdown had inverse effects in vitro and markedly decreased local and metastatic growth in vivo. XL184 abrogated human MPNST xenograft growth and metastasis in SCID mice. Conclusions: Informative prognosticators and novel therapies are crucially needed to improve MPNST management and outcomes. We show an important role for MET in MPNST, supporting continued investigation of novel anti-MET therapies in this clinical context. Clin Cancer Res; 17(12); 3943–55. ©2011 AACR.

Midkine Enhances Soft-Tissue Sarcoma Growth: A Possible Novel TherapeuticTarget

Purpose: New therapeutic targets for soft-tissue sarcoma (STS) treatment are critically needed. Midkine (MK), a multifunctional cytokine, is expressed during midgestation but is highly restricted in normal adult tissues. Renewed MK expression was shown in several malignancies where protumorigenic properties were described. We evaluated the expression and function of MK in STS. Experimental Design: Immunohistochemistry, reverse transcription-PCR, and Western blotting (WB) evaluated MK expression in human STS tissues and cell lines. WB and flow cytometry analyzed MK receptor expression. Cell growth assays evaluated the effect of MK on STS cell growth, and WB assessed MK downstream signaling. MK knock-in and knockout experiments further evaluated MK function. The growth of parental versus MK-transfected human fibrosarcoma cells was studied in vivo. Results: MK was found to be overexpressed in a variety of human STS histologies. Using a rhabdomyosarcoma (RMS) tissue microarray, cytoplasmic and nuclear MK was identified; nuclear MK expression was significantly increased in metastases. Similarly, several STS cell lines expressed and secreted MK; RMS cells exhibited nuclear MK. STS cells also expressed the MK receptors protein tyrosine phosphatase ζ and lipoprotein receptor-related protein. MK significantly enhanced STS cell growth potentially via the Src and extracellular signal-regulated kinase pathways. STS cells stably transfected with MK exhibited increased growth in vitro and in vivo. MK-expressing human STS xenografts showed increased tumor-associated vasculature. Furthermore, MK knockdown resulted in decreased STS cell growth, especially in RMS cells. Conclusion: MK enhances STS tumor growth; our results support further investigation of MK and its receptors as therapeutic targets for human STS.

Combined Vascular Endothelial Growth Factor Receptor/Epidermal Growth Factor Receptor Blockade with Chemotherapy for Treatment of Local, Uterine, and Metastatic Soft Tissue Sarcoma

Purpose: Soft tissue sarcoma (STS) is a rare heterogeneous malignancy. Overall survival has been stagnant for decades, primarily because systemic therapies are ineffective versus metastases, the leading cause of STS lethality. Consequently, we examined whether tyrosine kinase receptors active in STS growth signaling might be blockable and whether multireceptor blockade might synergize with low-dose STS chemotherapy by therapeutically affecting STS cells and their associated microenvironment. Experimental Design: Vandetanib (AstraZenca), a tyrosine kinase inhibitor of vascular endothelial growth factor receptor 2 and epidermal growth factor receptor, was evaluated alone and with chemotherapy in vitro and in vivo in three human STS nude mouse xenograft models of different STS locations (muscle, uterus, lung), stages (primary, metastatic), and subtypes (leiomyosarcoma, fibrosarcoma, uterine sarcoma: luciferase-expressing MES-SA human uterine sarcoma cells surgically implanted into uterine muscularis with bioluminescence tumor growth assessment; developed by us). Results:In vitro, human STS cells were sensitive to vandetanib. Vandetanib alone and with chemotherapy statistically significantly inhibited leiomyosarcoma local growth and fibrosarcoma lung metastasis. Direct injection of MES-SA into nude mice uterine muscularis resulted in high tumor take (88%), whereas s.c. injection resulted in no growth, suggesting microenvironmental tumor growth modulation. Vandetanib alone and with chemotherapy statistically significantly inhibited uterine sarcoma growth. In all models, vandetanib induced increased apoptosis, decreased tumor cell proliferation, and decreased angiogenesis. Conclusions: Vandetanib has antitumor effects against human STS subtypes in vitro and in vivo, where it also affects the tumor-associated microenvironment. Given the urgent need for better systemic approaches to STS, clinical trials evaluating vandetanib, perhaps with low-dose chemotherapy, seem warranted.

Establishment and characterization of a new human myxoid liposarcoma cell line (DL-221) with the FUS-DDIT3 translocation

Myxoid liposarcoma has the pathognomonic fusion oncogene FUS-DDIT3 encoding a chimeric transcription factor. Metastatic risk is higher with an increased round cell component and has been linked to aberrations involving the IGFR/PI3K/AKT pathway. These molecular insights have yet to translate to targeted therapies, and the lack of experimental models is a major hindrance. We describe the initial in-depth characterization of a new cell line (DL-221) and establishment of a mouse xenograft model. The cell line DL-221 was derived from a metastatic pleural lesion showing myxoid and round cell histology. This newly established cell line was characterized for phenotypic properties and molecular cytogenetic profile, using PCR, COBRA-FISH, and western blot. Next-generation whole-exome sequencing was performed to further characterize the cell line and the parent tumor. NOD-SCID-IL2R gamma knockout mice were xenograft hosts. DL-221 cells grew an adhering monolayer and COBRA-FISH showed an aneuploid karyotype with t(12;16)(q13;p11) and several other rearrangements; RT-PCR demonstrated a FUS-DDIT3 fusion transcript type 1. Both the cell line and the original tumor harbored a TP53 compound heterozygous mutation in exon 4 and 7, and were wild-type for PIK3CA. Moreover, among the 1254 variants called by whole-exome sequencing, there was 77% concordance between the cell line and parent tumor. The recently described hotspot mutation in the TERT promoter region in myxoid liposarcomas was also found at C228T in DL-221. Xenografts suitable for additional preclinical studies were successfully established in mice after subcutaneous injection. The established DL-221 cell line is the only published available myxoid liposarcoma cell line that underwent spontaneous immortalization, without requiring SV40 transformation. The cell line and its xenograft model are unique and helpful tools to study the biology and novel potential-targeted treatment approaches for myxoid liposarcoma.

Abstract 4484: The dual PI3K/mTOR inhibitor, PI-103, demonstrates therapeutic efficacy in undifferentiated pleomorphic sarcoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The lack of effective systemic therapy is the major unresolved clinical problem in undifferentiated pleomorphic sarcoma (UPS)/malignant fibrohistocytoma (MFH). Novel effective therapeutic approaches are crucially needed. Given the heterogeneity of UPS/MFH, identifying common molecular deregulations amenable to therapeutic targeting would be of major importance. PI3K/AKT/mTOR pathway has been implicated in cancer progression and metastasis, and is highly activated in soft tissue sarcoma (STS). However, not much is known about this pathway in UPS/MFH specifically. We sought to assess AKT/mTOR pathway expression and activation in human UPS/MFH specimens and to evaluate whether combined inhibition of this pathway affects the proliferation and survival of UPS/MFH cells. Materials and methods: A UPS/MFH tissue microarray (TMA) consisting of 180 primary UPS/MFH samples from patients who underwent complete surgical resection has been used to detect the expression of phospho-AKT. Human UPS/MFH primary cell strains/cell lines and murine undifferentiated pleomorphic sarcoma cell lines have been established to determine the pro-tumorigenic effects of PI3K/AKT/mTOR intracellular signaling and to evaluate the efficacy of dual PI3K/mTOR inhibitor PI-103 on cell growth and survival. Results: Phospho-AKT was found to be commonly expressed in UPS/MFH. Enhanced pAKT expression levels inversely correlated with shorter 5-yr disease specific survival (DSS). Phosphorylation of AKT, p70S6K and 4E-BP1 were observed in a panel of human UPS/MFH primary cell strains/cell lines and murine UPS cell lines. PI-103 inhibited PI3/AKT/mTOR pathway signaling in UPS/MFH as observed by WB analysis resulting in marked decrease in tumor cell proliferation, enhanced G1 phase cell cycle arrest, and abrogated migration and invasion. Interestingly, PI103 treatment resulted in decreased expression and activation of the tyrosine kinase receptors EGFR and c-MET. This novel finding suggests that a feedback loop may exist between AKT/mTOR and tyrosine kinase receptor expression. Conclusions: PI3K/AKT/mTOR pathway is a common molecular deregulation in the heterogeneous group of UPS/MFH. The Dual PI3K/mTOR inhibitor, PI-103, should be further be investigated for its anti-UPS/MFH effects. 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 4484. doi:10.1158/1538-7445.AM2011-4484

Nasopharyngeal carcinoma outcome with induction chemotherapy followed by concurrent chemoradiotherapy

Treatment of Nasopharyngeal Carcinoma (NPC) has been based on the Intergroup 0999 trial with chemoradiation (CRT) and consolidation chemotherapy (CT). While effective, toxicities are significant. As a result, many oncologists use induction chemotherapy (IC) followed by CRT, citing better tolerance with anecdotally no worse outcome. We reviewed 95 NPC patients treated between 2005 and 2015 at MDACC with IC followed by CRT. Median age was 49 years. Fifty-seven were T3/T4 and 62 were N2-3. The most common IC regimen was a platinum-taxane doublet (N = 72). 83 patients completed IC. Grade 3-4 toxicities with IC occurred in 10 patients. There were 15 primary site complete responses (CR), 68 partial responses (PR),6 stable disease (SD), and 2 progressed. There were 10 nodal CR, 73 PR, 4 SD, and 3 progressed. 92 patients received RT, 74 with CRT. At completion of treatment, there were 81 CR and 8 PR patients. Post radiation toxicities included mucositis and skin rash (37), oto- toxicity (25), PEG placement (12), and osteonecrosis (2). Three-year progression free survival (PFS) and distant metastasis free survival (DMFS) were 77.3% and 78%. CRT for advanced NPC is standard, but IC remains controversial. Early trials failed to show a benefit but used older chemotherapy and pre-intensity modulated radiation therapy (IMRT) methods. Modern trials with platinum-taxane regimens and IMRT have shown reasonable PFS and OS results with acceptable toxicity. This retrospective review of IC followed by CRT showed acceptable toxicity and good response and survival outcomes. This approach has, for many oncologists, become a standard.

Clinical outcomes after local field conformal reirradiation of patients with retropharyngeal nodal metastasis

The purpose of this study was to present our experience with retropharyngeal node reirradiation using highly conformal radiotherapy (RT).

Abstract 4577: Targeting N-glycosylation of tyrosine kinase receptors for preventing tumorigenesis and malignant transformation of neurofibromatosis type I

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Neurofibromatosis type I (NF1) is a dominantly inherited disease affecting 1 in every 2,500 to 3,000 individuals, representing the most common familial cancer predisposition syndrome. The hallmark of clinical manifestation of NF1 is the development of multiple neurofibromas, which are highly heterotypic benign tumors of peripheral nerve sheath mainly composed of immature Schwann cells, fibroblasts, perineurial and inflammatory matrix. NF1 patients are also at high risk for the development of certain maligancies such as pheochromocytomas, childhood myeloid leukemias, neuroblastomas, rhabdomyosarcomas, and malignant peripheral nerve sheath tumors (MPNSTs). Because of the potential involvement of underlying nerves and blood vessels, surgical removal of tumors is not always an option. Once progressing to MPNSTs, although the resection is possible, most patients will eventually relapse locally or systemically. There is no effective treatment for NF1, nor effective approaches for predicting or preventing the occurrence of devastating complications. Thus, to develop agents for preventing or reversing the tumorigenesis and malignant transformation of NF1 are critically needed. In this report, we found HGF, c-MET, EGF and EGFR are aberrantly expressed in clinical specimens of neurofibromas and MPNSTs with NF and that autocrine and/or paracrine HGF/c-MET and EGF/EGFR loops promote MPSNT cell migration and invasion in vitro (Fig1). We also found that the loss of Nf1 in neurofibromas and MPNSTs results in hyperactive of Ras (Fig2). Compared with normal Schwann cells, MPNST cells express more N-glycoproteins and display different N-glycoprotein signatures (Fig3). In order to confirm our hypothesis, we treated cells with Tunicamycin, or 2-DG (N-glycosylation inhibitors) respectively in variable doses for different time. We found that both Tunicamycin and 2-DG inhibited the proliferation and promoted apoptosis of MPNST cell lines but had no effect on normal human Schwann cells (Fig4); inhibited the glycosylation and phosphorylation of EGFR and c-MET and impaired receptor-mediated MEK-ERK1/2 and PI3K-AKT signaling in a dose-dependent manner (Fig5). Moreover, 2-DG inhibited the maturation of c-MET as demonstrated by the intact premature chain, failing to cleave into one mature α-chain and another mature α-chain. In addition, 2-DG inhibited the translocation of the receptors from the cytoplasm onto the cell surface and retained receptors in the ER and Golgi apparatus (Fig6). To date, 2-DG inhibition of N-glycosylation and phosphorylation of tyrosine kinase receptors has not been reported. These novel findings prompted us to explore whether 2-DG disrupts Ras-directed N-glycosylation of tyrosine receptors in Schwann cell progenitors and tumor cells and to evaluate its roles in preventing the tumorigenesis and malignant transformation of NF1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4577. doi:1538-7445.AM2012-4577