Co-targeting PI3K, mTOR, and IGF1R with small molecule inhibitors for treating undifferentiated pleomorphic sarcoma.

Undifferentiated pleomorphic sarcomas (UPSs) are aggressive mesenchymal malignancies with no definitive cell of origin or specific recurrent genetic hallmarks. These tumors are largely chemoresistant; thus, identification of potential therapeutic targets is necessary to improve patient outcome. Previous studies demonstrated that high expression of activated protein kinase B (AKT) in patients with UPS corresponds to poor disease-specific survival. Here, we demonstrate that inhibiting phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling using a small molecule inhibitor reduced UPS cell proliferation and motility and xenograft growth; however, increased phosphorylation of insulin-like growth factor 1 receptor (IGF1R) indicated the potential for adaptive resistance following treatment through compensatory receptor activation. Co-treatment with a dual PI3K/mTOR inhibitor and an anti-IGF1R kinase inhibitor reduced in vivo tumor growth rates despite a lack of antiproliferative effects in vitro. Moreover, this combination treatment significantly decreased UPS cell migration and invasion, which is linked to changes in p27 subcellular localization. Our results demonstrate that targeted inhibition of multiple components of the IGF1R/PI3K/mTOR pathway was more efficacious than single-agent therapy and suggest that co-targeting this pathway could be a beneficial therapeutic strategy for patients with UPS.

Analysis of Clinical and Molecular Factors Impacting Oncologic Outcomes in Undifferentiated Pleomorphic Sarcoma.

BACKGROUND: Undifferentiated pleomorphic sarcomas (UPS) present a diagnostic and therapeutic challenge. Identification of prognostic molecular markers is required for the discovery of novel treatment approaches. The purpose of this study was to correlate clinicopathologic variables, expression of tyrosine kinase receptors, and markers of cell cycle progression and survival with oncologic outcomes. METHODS: A tissue microarray containing 208 primary UPS samples was analyzed by immunohistochemistry for protein markers and in situ hybridization for microRNA. Staining results were correlated with clinicopathologic features and oncologic outcomes. Univariate and multivariate analyses were conducted to assess associations between expression of protein markers, mi-RNA, and outcome. RESULTS: At a median follow-up of 3.9 years (9 years for survivors), 5-year disease-specific survival (DSS) was 63 %. Clinical variables associated with improved DSS included age <61 years, tumor size <10 cm, margin-negative resection, and sporadic-tumor status. At the protein level, loss of cyclin D1 (p = 0.06), pEGFR (p = 0.023), pIGF-1R (p = 0.022), and PTEN (p < 0.001) and overexpression of AXL (p = 0.015) were associated with reduced DSS on univariate analysis. Ki67, PCNA, and pEGFR were more highly expressed in sporadic UPS than radiation-associated (RA-UPS), whereas RA-UPS samples expressed higher levels of both phosphorylated and total IGF-1R. DISCUSSION: Loss of cyclin D1, overexpression of AXL, and loss of PTEN are associated with poor cancer-specific outcomes and warrant further investigation in UPS. The differences in protein expression in sporadic versus RA-UPS may indicate that the activated molecular signaling nodes may be different for each specific histology and also could explain the aggressive phenotype seen in RA-UPS compared with the sporadic lesions.

Clinical Observations and Molecular Variables of Primary Vascular Leiomyosarcoma.

IMPORTANCE: Vascular leiomyosarcomas are a rare subtype of leiomyosarcomas that most commonly affect the inferior vena cava and account for 5% of all leiomyosarcomas. These tumors are aggressive malignant tumors for which adjuvant modalities have not shown increased efficacy compared with surgery. OBJECTIVES: To evaluate the outcomes of patients with vascular leiomyosarcoma and the association between vascular leiomyosarcomas and immunohistochemical molecular markers, to determine their potential prognostic and therapeutic utility. DESIGN, SETTING, AND PARTICIPANTS: Retrospective medical record review of a cohort of 77 patients who presented to the University of Texas MD Anderson Cancer Center in Houston during the period from January 1993 to April 2012. Data were analyzed during the period from November 2012 to May 2015. All of the patients received a confirmed diagnosis of vascular leiomyosarcoma. Immunohistochemical studies for biomarkers were performed on a tissue microarray that included 26 primary specimens of vascular leiomyosarcoma. MAIN OUTCOMES AND MEASURES: Demographic and clinical factors were evaluated to assess clinical course, patterns of recurrence, and survival outcomes for patients with primary vascular leiomyosarcoma. A univariate Cox proportional hazards model was used to correlate disease-specific survival and time to recurrence with potential prognostic indicators. RESULTS: Sixty-three patients with localized disease who underwent surgical resection formed the study population, and their data were used for subsequent outcomes analysis. The median age at diagnosis was 58 years (range, 22-78 years). The majority of patients were female (41 patients [65%]) and white (51 patients [81%]). The 5-year disease-specific survival rate after tumor resection was 65%. The median time to local recurrence was 43 months, the median time to distant recurrence was 25 months, and the median time to concurrent local and distant recurrences was 15 months (P = .04). Strong expressions of cytoplasmic ß-catenin (hazard ratio, 5.33 [95% CI, 0.97-29.30]; P = .06) and insulinlike growth factor 1 receptor (hazard ratio, 2.74 [95% CI, 1.14-6.56]; P = .02) were associated with inferior disease-specific survival. CONCLUSIONS AND RELEVANCE: Vascular leiomyosarcomas are aggressive malignant tumors, with high recurrence rates. Expressions of ß-catenin and insulinlike growth factor 1 receptor were associated with poor disease-specific survival. Prospective studies should evaluate the clinical and therapeutic utility of these molecular markers.

Poly (ADP) ribose polymerase inhibition: A potential treatment of malignant peripheral nerve sheath tumor.

Poly (ADP) ribose polymerase (PARP) inhibitors, first evaluated nearly a decade ago, are primarily used in malignancies with known defects in DNA repair genes, such as alterations in breast cancer, early onset 1/2 (BRCA1/2). While no specific mutations in BRCA1/2 have been reported in malignant peripheral nerve sheath tumors (MPNSTs), MPNST cells could be effectively targeted with a PARP inhibitor to drive cells to synthetic lethality due to their complex karyotype and high level of inherent genomic instability. In this study, we assessed the expression levels of PARP1 and PARP2 in MPNST patient tumor samples and correlated these findings with overall survival. We also determined the level of PARP activity in MPNST cell lines. In addition, we evaluated the efficacy of the PARP inhibitor AZD2281 (Olaparib) in MPNST cell lines. We observed decreased MPNST cell proliferation and enhanced apoptosis in vitro at doses similar to, or less than, the doses used in cell lines with established defective DNA repair genes. Furthermore, AZD2281 significantly reduced local growth of MPNST xenografts, decreased the development of macroscopic lung metastases, and increased survival of mice with metastatic disease. Our results suggest that AZD2281 could be an effective therapeutic option in MPNST and should be further investigated for its potential clinical use in this malignancy.

AXL is a potential therapeutic target in dedifferentiated and pleomorphic liposarcomas.

BACKGROUND: AXL is a well-characterized, protumorigenic receptor tyrosine kinase that is highly expressed and activated in numerous human carcinomas and sarcomas, including aggressive subtypes of liposarcoma. However, the role of AXL in the pathogenesis of well-differentiated (WDLPS), dedifferentiated (DDLPS), and pleomorphic liposarcoma (PLS) has not yet been determined. METHODS: Immunohistochemical analysis of AXL expression was conducted on two tissue microarrays containing patient WDLPS, DDLPS, and PLS samples. A panel of DDLPS and PLS cell lines were interrogated via western blot for AXL expression and activity and by ELISA for growth arrest-specific 6 (GAS6) production. AXL knockdown was achieved by siRNA or shRNA. The effects of AXL knockdown on cell proliferation, migration, and invasion were measured in vitro. In addition, AXL shRNA-containing DDLPS cells were assessed for their tumor-forming capacity in vivo. RESULTS: In this study, we determined that AXL is expressed in a subset of WDLPS, DDLPS, and PLS patient tumor samples. In addition, AXL and its ligand GAS6 are expressed in a panel of DDLPS and PLS cell lines. We show that the in vitro activation of AXL via stimulation with exogenous GAS6 resulted in a significant increase in cell proliferation, migration, and invasion in DDLPS and PLS cell lines. Transient knockdown of AXL resulted in attenuation of these protumorigenic phenotypes in vitro. Stable AXL knockdown not only decreased migratory and invasive characteristics of DDLPS and PLS cells in vitro but also significantly diminished tumorigenicity of two dedifferentiated liposarcoma xenograft models in vivo. CONCLUSIONS: Our results suggest that AXL signaling contributes to the aggressiveness of DDLPS and PLS, and that AXL is therefore a potential therapeutic target for treatment of these rare, yet devastating tumors.

The hepatocyte growth factor receptor as a potential therapeutic target for dedifferentiated liposarcoma.

Dedifferentiated liposarcomas (DDLPS) are highly resistant to conventional chemo- and radiotherapies, with surgical resection remaining the classic treatment strategy; therefore, there is a pressing need for novel anti-DDLPS-targeted chemotherapeutics. Hepatocyte growth factor receptor (Met) expression is elevated in DDLPS, but the functional role of Met signaling in this disease is not known. We found that the in vitro stimulation of DDLPS cells with hepatocyte growth factor (HGF) elevated the degree of PI3K/AKT and MAPK pathway signaling, and that pro-tumorigenic phenotypes such as cell proliferation, invasion, and migration were significantly enhanced. Conversely, Met knockdown using shRNA-mediated interference decreased HGF-induced Met signaling, the invasive and migratory nature of DDLPS cells in vitro, and the tumorigenicity of DDLPS cells in vivo. These data strongly support the role for Met as a DDLPS therapeutic target. To that end, using EMD1214063, an ATP-competitive kinase inhibitor that targets Met more specifically than other kinases, inhibited Met-dependent signaling, reduced the oncogenicity of DDLPS cells in vitro, and significantly increased the survival of nude mice bearing subcutaneous DDLPS xenografts. These findings support further investigations of HGF-induced Met signaling inhibition in DDLPS, as a potential strategy to enhance clinical outcomes for this disease.

Long-term outcomes in patients with radiation-associated angiosarcomas of the breast following surgery and radiotherapy for breast cancer.

BACKGROUND: Radiation-associated angiosarcoma (RAAS) is a devastating disease occasionally observed in breast cancer patients treated with radiation. Due to its rarity, our knowledge-of disease risk factors, epidemiology, treatment, and outcome-is extremely limited. Therefore, we sought to identify clinicopathologic factors associated with local and distant recurrence and disease-specific survival (DSS). METHODS: Radiation-associated angiosarcoma was defined as pathologically confirmed breast or chest wall angiosarcoma arising within a previously irradiated field. A comprehensive search of our institutional tumor registry (1/1/93 through 2/28/11) was used to identify patients (n = 95 females). Patient, original tumor, RAAS treatment, and outcome variables were retrospectively retrieved and assembled into a database. RESULTS: The median follow-up for all RAAS patients was 10.3 (range, 2.4-31.8) years. The latency period following radiation exposure ranged from 1.4 to 26 (median, 7) years. One-year and 5-year DSS rates were 93.5 and 62.6 %, respectively. Reduced risk of local recurrence was observed in patients who received chemotherapy (P = 0.0003). In multivariable analysis, size was found to be an independent predictor of adverse outcome (P = 0.015). CONCLUSIONS: Our study demonstrates that RAAS exhibits high recurrence rates. It also highlights the need for well-designed, multicenter, clinical trials to inform the true utility of chemotherapy in this disease.

High-resolution analysis of four efficient yeast replication origins reveals new insights into the ORC and putative MCM binding elements.

In budding yeast, the eukaryotic initiator protein ORC (origin recognition complex) binds to a bipartite sequence consisting of an 11 bp ACS element and an adjacent B1 element. However, the genome contains many more matches to this consensus than actually bind ORC or function as origins in vivo. Although ORC-dependent loading of the replicative MCM helicase at origins is enhanced by a distal B2 element, less is known about this element. Here, we analyzed four highly active origins (ARS309, ARS319, ARS606 and ARS607) by linker scanning mutagenesis and found that sequences adjacent to the ACS contributed substantially to origin activity and ORC binding. Using the sequences of four additional B2 elements we generated a B2 multiple sequence alignment and identified a shared, degenerate 8 bp sequence that was enriched within 228 known origins. In addition, our high-resolution analysis revealed that not all origins exist within nucleosome free regions: a class of Sir2-regulated origins has a stably positioned nucleosome overlapping or near B2. This study illustrates the conserved yet flexible nature of yeast origin architecture to promote ORC binding and origin activity, and helps explain why a strong match to the ORC binding site is insufficient to identify origins within the genome.

Epithelial-mesenchymal transition and cancer stem cells: a dangerously dynamic duo in breast cancer progression.

Aberrant activation of a latent embryonic program - known as the epithelial-mesenchymal transition (EMT) - can endow cancer cells with the migratory and invasive capabilities associated with metastatic competence. The induction of EMT entails the loss of epithelial characteristics and the de novo acquisition of a mesenchymal phenotype. In breast cancer, the EMT state has been associated with cancer stem cell properties including expression of the stem cell-associated CD44+/CD24-/low antigenic profile, self-renewal capabilities and resistance to conventional therapies. Intriguingly, EMT features are also associated with stem cells isolated from the normal mouse mammary gland and human breast reduction tissues as well as the highly aggressive metaplastic and claudin-low breast tumor subtypes. This has implications for the origin of these breast tumors as it remains unclear whether they derive from cells that have undergone EMT or whether they represent an expansion of a pre-existing stem cell population that expresses EMT-associated markers to begin with. In the present review, we consider the current evidence connecting EMT and stem cell attributes and discuss the ramifications of these newly recognized links for our understanding of the emergence of distinct breast cancer subtypes and breast cancer progression.