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Malignant Fibrous Histiocytoma (MFH) in the Sarcoma Learning Center

Targeting the PI3K/AKT Pathway in UPS/MFH :
Aiming Towards Novel Therapy

An ESUN Article

Quansheng Zhu, MD, PhD
Department of Surgical Oncology
and
Dina Chelouche Lev, MD
Department of Cancer Biology
The University of Texas M.D. Anderson Cancer Center
Houston, Texas

Introduction to UPS/MFH

Unclassified pleomorphic sarcomas (UPS) represent a major diagnostic and most importantly a therapeutic challenge. The history of this soft tissue sarcoma subset dates to the 1960’s when it was first defined as malignant fibrous histocytoma (MFH). Thirty years later this terminology was refuted demonstrating that MFH truly represent a cluster of poorly differentiated malignancies of heterogeneous origin (both epithelial and mesenchymal) sharing a similar morphological pattern; a large proportion of tumors originally diagnosed as MFH could be re-classified using electron microscopy evaluation, immunohistochemical analysis, and molecular genetics. With this highly clinically relevant paradigm shift, sarcoma caregivers are still confronted by a subset of soft tissue sarcomas exhibiting pleomorphic morphology that can not be further classified. To that end the 2002 WHO classification recognized the existence of an undifferentiated category of pleomorphic sarcoma, which was termed UPS/MFH[1]. Clinically, UPS/MFH exhibit peak incidence in the 6th and 7th decades of life, show no gender predilection, and occur mainly in the deep soft tissues of the extremities and trunk[2]. Therapy for localized disease consists of surgery ± radiotherapy. However, 30-50% local recurrence rates (depending on tumor site) remain problematic and are particularly ominous in loci where salvage radical surgery may not be feasible. Systemic failure, mainly lung metastasis, is the major determinant of poor patient outcome and current chemotherapies are of only minimal impact [3-4]. Thus, there is a crucial need for novel anti-UPS/MFH effective therapeutic approaches.

The AKT pathway as a novel therapeutic target

Molecularly-targeted therapy has emerged as the new anti-cancer paradigm with the hope of more selectively impacting cancer cells rather than normal cells, thus potentially minimizing treatment-related morbidities. This approach has led to recent advances in several diseases, including GIST [5], an STS subtype. Effective targeted therapy requires an accessible and functional target. To utilize targeted therapy for UPS/MFH, enhanced knowledge of potential targets and their role in UPS/MFH progression and metastasis are needed. In light of UPS/MFH genetic complexity and molecular heterogeneity, identifying a common molecular deregulation amenable for therapeutic targeting is highly important. AKT kinase, known also as protein kinase B (PKB), a serine-threonine kinase discovered originally as the cellular homolog of the v-AKT oncogene[6] , is a convergence point for several extracellular and other upstream signals [Figure 1].

Figure 1: AKT-mediated intracellular signaling

AKT activation functions as a master switch to generate a plethora of intracellular signals and intracellular responses. AKT kinase is activated by phosphorylation on two critical residues: threonine 308 (T308) within the activation loop, and serine 473 (S473) at the C-terminal portion of the protein. AKT activation is mediated by PI3 kinase which in turn is activated by several cell surface receptors and cognate molecules [7-8], while negative regulation of PI3K-dependent AKT activation occurs via tumor suppressor genes such as PTEN and SHIP phosphatases[9]. Once activated, AKT induces multiple downstream pathways, promoting proliferation and increased cell survival. Multiple mechanisms may contribute to cancer-associated AKT hyperactivation, including amplifications, mutations and/or aberrant overexpression and activation of upstream tyrosine kinase receptors.

Considerable evidence supports a key AKT role in human cancer. For example, AKT activation has been demonstrated in brain, prostate, breast, lung, liver, gastric, colon, ovarian, and endometrial cancers and has been implicated in cancer progression and metastasis[9]. While not extensively explored in soft tissue sarcoma (STS), recent evidence suggests AKT pathway involvement in complex karyotype STS development and progression[10]. Hernando et al reported increased expression of activated AKT in a large panel of human leiomyosarcoma, UPS/MFH, and dedifferentiated liposarcoma ; using a conditional PTEN knockout mouse model, a critical role for the AKT pathway in smooth muscle transformation and leiomyosarcoma development was shown. Tomita et al identified a correlation between phospho-AKT (pAKT) expression in human STS specimens, subsequent tumor recurrence, and patient survival[11].

Recently we showed that pAKT is highly expressed in a diverse cohort of human STS histological subtypes (although UPS/MFH were not included), where it serves as a convergence point for many upstream deregulations. Furthermore, we have demonstrated that AKT inhibition significantly impacts STS cell proliferation, cell cycle inhibition and apoptosis in vitro as well as STS growth in vivo [12]. Together, these results suggest a possible role for AKT activation in STS. Further studies in UPS/MFH will hopefully further illuminate the function of AKT and most importantly, the possible yield of AKT inhibitors as a novel therapy for this STS histological subset.

Hypothesis and specific aims

Based on previously published data and our own preliminary studies we hypothesize that AKT activation is a potential common deregulation in UPS/MFH and that AKT inhibition can abrogate tumor and metastasis growth. To that end, we propose to evaluate the impact of AKT blockade on UPS/MFH pro-tumorigenic and pro-metastatic processes. The following two Specific Aims are suggested:

• Aim 1: To evaluate the effect of AKT inhibition on UPS/MFH cell growth, survival, migration, and invasion. Effects of targeting specific AKT isoforms will be tested separately.
• Aim 2: To examine the effect of AKT inhibition on UPS/MFH growth and metastasis in vivo.

UPS/MFH Bioresources and Study Design

The major obstacle for comprehensive “bed-side to bench” UPS/MFH translational studies is the relative lack of requisite bioresources; i.e., tumor specimens, cell lines and animal models. To that end we have established a clinically annotated human UPS/MFH tissue microarray (~180 primary UPS/MFH specimens) that can enable us to evaluate the correlation between marker expression and patient outcome.

Tissue microarrays (TMAs) consist of paraffin blocks in which numerous separate tissue cores are assembled in array fashion to allow multiplexed histological analysis of biomarkers. Advantages of this technology include conservation of valuable tissue samples and uniform treatment of all samples analyzed in a study. When linked to clinical follow-up data, TMAs are powerful tools for biomarker discovery and analysis.

A UPS/MFH primary cell culture bank has also been created and currently contains more than 15 fresh surgical specimen-derived UPS/MFH cell strains available for in vitro studies; all have been characterized cytogenetically and shown to grow in soft agar. These cells harbor complex karyotypes and several reproducibly result in tumor growth when injected into SCID mice.

As part of the current proposal gene expression profiling of cell lines/strains and original tumors will be conducted using the illumina platform to determine the applicability of their utilization as UPS/MFH models. Screening several of these cell strains we have recently confirmed high pAKT expression levels. Moreover, through collaboration (Dr David Kirsch, Duke University) we have recently acquired a temporally restricted STS mouse model[13] where intramuscular injection of adenovirus expressing Cre recombinase (Ad-Cre) into the extremities of mice (mixed S4/SvJae and C57/Bl6) with conditional mutations of Kras and Trp53 (both are very common STS genetic aberrations) induces the development of high grade sarcoma resembling UPS/MFH in more than 90% of mice after a median time of 3 months. Once tumor is established it will grow to 1.5cm within 3-4 weeks; at this time 20% of mice will exhibit spontaneous microscopic lung metastasis. Furthermore, if more time is allowed for metastasis growth via resection of the primary tumor, upon local recurrence (occurring in 100% of mice within several weeks), ~50% of mice will exhibit lung metastasis which have been confirmed histologically to be sarcomas. This model optimally mirrors human UPS/MFH clinical behavior and is therefore a highly relevant system in which to assess novel therapies.

Utilizing these unique bioresources experiments will be conducted towards the completion of both study Aims. Specifically, AKT blockade will be induced using synthetic inhibitors as well as siRNA knockdown of each one of the different three AKT isoforms and effect on tumor cell growth, migration, invasion and survival which will be evaluated utilizing appropriate assays, respectively. Effect on AKT downstream target expression and phosphorylation will be assessed via western blotting and kinase assays. The expression of AKT downsteam targets in human samples and their correlation with patient clinical outcome will be evaluated using the UPS/MFH TMA. Furthermore, in vitro evaluations will be complimented by in vivo testing of the effect of AKT blockade on tumor and metastasis growth using the models described above.

Cre recombinase (Cre) is a Type I topoisomerase from P1 bacteriophage that catalyzes site-specific recombination of DNA between loxP sites. The loxP recognition element is a 34 base pair (bp) sequence composed of two 13 bp inverted repeats flanking an 8 bp spacer region that confers directionality. In a nutshell, Cre is a valuable tool to manipulate genes and chromosomes. It is used to generate animals with mutations limited to certain cell types (tissue-specific knockout) or animals with mutations that can be activated by drug administration (inducible knockout). The availability of transgenic animal with tissue specific or inducible Cre expression permits researchers to inactivate or activate a gene of interest simply by breeding a floxed animal to pre-existing Cre-transgenics. These techniques offer unprecidented experimental control of genetic manipulation and the ability to do tightly controlled experiements not possible in earlier generation transgenic animals.

Significance

There is a crucial need for more efficacious therapeutic strategies to improve the outcome of patients suffering from UPS/MFH. Extensive studies of the AKT signaling pathway in a variety of epithelial tumors suggests it to be an attractive molecular target for cancer therapy, leading to the development of several AKT specific inhibitors now ready for clinical trials. Based on limited previously published studies and our own preliminary data, it is possible that AKT-mediated intracellular signaling might be relevant in UPS/MFH progression. Studies proposed here will enable validation of these initial insights while also further evaluating the potential efficacy of AKT as an anti-UPS/MFH therapeutic target. Positive findings have the potential for significant impact on the management and outcome of patients suffering from UPS/MFH.

Editor's Note: This study is funded by a $50,000 grant from the Liddy Shriver Sarcoma Initiative.

References

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