Study Report: Exploitation of the Mammalian Hippo Pathway and its Members in the Treatment of Alveolar Rhabdomyosarcoma

By Lisa Crose, PhD and Corinne Linardic MD, PhD

Introduction

Rhabdomyosarcomas (RMS) are malignancies of skeletal muscle histogenesis that are the most common childhood soft tissue sarcomas. While there are several histologic variants, the most common are embryonal (eRMS) and alveolar RMS (aRMS). The aRMS subtype is the most difficult to cure, with a 5-year survival of <50%.² Many aRMS tumors bear a signature genetic change resulting in the expression of the PAX3-FOXO1 fusion gene. When PAX3-FOXO1 is expressed, it reactivates pro-proliferative embryonic muscle signaling pathways. Children with metastatic, PAX3-FOXO1-positive aRMS have the worst prognosis, with a 5-year survival of <10%. These high risk patients have few therapeutic options; therefore, the goal of our laboratory is to identify new drug targets tailored to the genetic changes associated with the disease.

An important tool for determining the role of genetic changes in tumorigenesis is in vivo modeling. Our laboratory has taken the approach of modeling aRMS from primary (non-malignant) human skeletal muscle myoblasts (HSMMs). This is accomplished by the sequential expression in HSMMs of PAX3-FOXO1, followed by the catalytic subunit of telomerase (hTERT), followed by the MycN oncogene (model termed HSMM^PF+H+M). This approach transforms HSMMs in vitro and produces xenografts in vivo that histologically mimic human aRMS.³ This model allows us to study aRMS initiation and step-wise progression in human cells, and to perform large-scale screens of new aRMS therapeutic targets in vivo.

We had previously shown that early introduction of PAX3-FOXO1 contributes to HSMM bypass of cellular senescence and primes cells for aRMS tumorigenesis.⁴ We therefore hypothesized that transcriptional changes promoted by PAX3-FOXO1 cause this phenomenon. To examine the transcriptional changes regulated by PAX3-FOXO1, we performed transcriptional profiling analysis of primary HSMMs expressing PAX3-FOXO1.

The resulting dataset was ~2600 significantly changed genes in PAX3-FOXO1 – expressing cells. One of these is the Ras Association Domain Family member RASSF4. Upon further examination, we found that RASSF4 expression was markedly elevated in PAX3-FOXO1-positive human aRMS cells and tumors with elevated RASSF4 levels associated with worse clinical outcome. These data suggested to us that RASSF4 is a vital component of aRMS biology that had not been described previously.

To determine the role of RASSF4 in aRMS, we performed loss-of-function studies. Using RASSF4 shRNAs, we found that RASSF4 suppression caused a profound growth arrest in aRMS cells.

RASSF4-knockdown cells also displayed characteristics of senescence, including senescent morphology, beta-galactosidase staining, and p21 tumor suppressor induction, suggesting that RASSF4 upregulation in aRMS is critical to promote proliferation and senescence evasion.

We next generated a doxycycline (dox)-inducible system to conditionally prevent RASSF4 expression. This system induces RASSF4 knockdown in the presence of dox and allowed us to examine the role of RASSF4 in aRMS xenografts in vivo. Inducible knockdown of RASSF4 significantly reduced tumor volume over time, validating the pro-proliferative role of RASSF4 in vivo.

There is little known about the biological function of RASSF4 in general, and no information on its molecular function in aRMS. To determine the molecular mechanism that RASSF4 utilizes to promote cell proliferation, we performed immunoprecipitation (IP) experiments to identify RASSF4 protein binding partners. RASSF4 is considered a protein scaffold and contains a Ras-association (RA) domain and a carboxy terminal SARAH domain. These domains are predicted to associate with the Ras oncogene and the MST1 tumor suppressor, respectively. Although we did not observe association between RASSF4 and Ras in aRMS cells, we did see robust association between RASSF4 and the MST1 tumor suppressor. This association was dependent on the RASSF4 SARAH domain.

Purpose of investigation

We have discovered the RASSF4-MST1 complex to be a vital signaling node for aRMS. However, the signaling events downstream of this complex are unknown. We hypothesize that inactivation of the Hippo pathway is necessary for aRMS initiation and maintenance, while reactivation of the Hippo pathway may be a new aRMS therapeutic strategy. The studies below will begin to define the biology of Hippo signaling in aRMS.

Results

1: Determine how RASSF4 inhibits the Hippo pathway

At the beginning of our work, we had found that RASSF4 and MST1 exist in a protein complex. However, the effects of RASSF4 on MST1 signaling were not known. We examined the effects of RASSF4 expression on MST1 signaling by performing phospho-immunoblot analysis of proteins known to be downstream of MST1, namely LATS1 and MOB1. We were surprised to find that loss of RASSF4 expression by shRNA did not impact LATS1 phosphorylation. However, we did find that RASSF4 was preventing activation of downstream signaling to the MOB1 tumor suppressor. This was found to be specific to MST1 activity, since expression of kinase-inactive version of MST1 could reverse the phosphorylation of MOB1 induced by RASSF4 loss.

Since expressing a kinase-inactive MST1 could reverse the signaling effects of RASSF4 on MOB1, we then examined if expressing kinase-inactive MST1 could reverse the cellular RASSF4-knockdown phenotype. We were pleased to find that expression of MST1 K59R could reverse the cell cycle arrest and senescence induction caused by RASSF4, suggesting that regulation of MST1 signaling is a major role of RASSF4.

Therefore, we have found that the MOB1 tumor suppressor is downstream of the RASSF4-MST1 complex. Future studies will examine how loss of MOB1 phosphorylation regulates aRMS tumorigenesis.

2: Examine the requirement for YAP in aRMS tumorigenesis

To determine the expression levels of the terminal Hippo protein YAP in primary human tumors, we performed YAP immunohistochemistry on a RMS tissue microarray. We found that in both major RMS subtypes, YAP was significantly upregulated compared to normal muscle. This suggested to us that YAP was playing an important oncogenic role in RMS.

We then used YAP loss of function to determine the role of YAP in aRMS cells. Using YAP shRNA, we blocked YAP expression in aRMS cells. YAP-deficient aRMS cells were deficient in their proliferative capacity as measured by BrdU incorporation. Surprisingly, YAP-deficient cells also underwent senescence, similar to RASSF4 knockdown. However, when we attempted to rescue RASSF4 knockdown with YAP expression, YAP did not reverse the RASSF4-knockdown phenotype. This suggested that the senescence induced by RASSF4 knockdown and the senescence induced by YAP knockdown were not related. However, RASSF4-deficient cells were deficient in YAP expression, but we hypothesize this is due to the senescence induction caused by RASSF4 loss itself.

In conclusion, we have identified a unique signaling node in aRMS that promotes cell proliferation and senescence evasion. RASSF4, a PAX3-FOXO1 target gene, is upregulated in aRMS and prevents activation of the Hippo pathway at the level of MST1. RASSF4-MST1 inhibitory complexes block activation of signaling to the MOB1 tumor suppressor. Further, the YAP oncoprotein is highly expressed in both RMS subtypes, suggesting a role for the Hippo pathway in both eRMS and aRMS subtypes. YAP-deficient aRMS cells had a decreased proliferative capacity and underwent cellular senescence. Altogether, this study suggests that targeting the Hippo pathway either at the level of MST1 or YAP may be beneficial as a therapeutic strategy.

Please see our manuscript in the Journal of Clinical Investigation, supported in part by LSSI: http://www.jci.org/articles/view/67087.

By Lisa Crose, PhD
Department of Pediatrics
Duke University Medical Center in Durham, North Carolina

and Corinne Linardic MD, PhD
Departments of Pediatrics, Pharmacology and Cancer Biology
Duke University Medical Center in Durham, North Carolina

References

1. Crose, L. E., Galindo, K. A., Kephart, J. G., Chen, C., Fitamant, J., Bardeesy, N., Bentley, R. C., Galindo, R. L., Ashley Chi, J. T., and Linardic, C. M. (2014) Alveolar rhabdomyosarcoma-associated PAX3-FOXO1 promotes tumorigenesis via Hippo pathway suppression. J Clin Invest 124, 285-296.
2. Ognjanovic, S., Linabery, A. M., Charbonneau, B., and Ross, J. A. (2009) Trends in childhood rhabdomyosarcoma incidence and survival in the United States, 1975-2005. Cancer 115, 4218-4226.
3. Naini, S., Etheridge, K. T., Adam, S. J., Qualman, S. J., Bentley, R. C., Counter, C. M., and Linardic, C. M. (2008) Defining the cooperative genetic changes that temporally drive alveolar rhabdomyosarcoma. Cancer Res 68, 9583-9588.
4. Linardic, C. M., Naini, S., Herndon, J. E., 2nd, Kesserwan, C., Qualman, S. J., and Counter, C. M. (2007) The PAX3-FKHR fusion gene of rhabdomyosarcoma cooperates with loss of p16INK4A to promote bypass of cellular senescence. Cancer Res 67, 6691-6699..

Exploitation of the Mammalian Hippo Pathway and its Members in the Treatment of Alveolar Rhabdomyosarcoma

By Lisa Crose, PhD and Corinne Linardic MD, PhD

Background

Alveolar rhabdomyosarcoma (aRMS) is an aggressive pediatric malignancy of skeletal muscle with poor prognosis and few therapeutic options. We have uncovered a new tumor suppressor pathway that is silenced in aRMS. This pathway is known as the mammalian Hippo pathway. This appears to be occurring through PAX3-FOXO1-mediated expression of Hippo pathway inhibitors. We find that experimental reactivation of the Hippo pathway causes aRMS cell growth-arrest in vitro, and inhibits aRMS tumorigenesis in vivo. We predict that reactivation of the Hippo pathway will be a useful therapeutic strategy to exploit in pediatric and adult cancers.

Our goal is to better understand the mechanism through which Hippo signaling is silenced in aRMS. Completion of the proposed studies will identify Hippo pathway regulators important in aRMS, and provide a platform for designing new aRMS mouse models and therapeutic approaches.

Alveolar Rhabdomyosarcoma: The Clinical Problem

Rhabdomyosarcomas (RMS) are malignancies of the skeletal muscle lineage that are the most common soft tissue sarcomas of childhood and adolescence. While there are several histologic variants, including embryonal (eRMS) and alveolar RMS (aRMS), aRMS is the most difficult to cure, with a 5-year survival of <50%.¹ Many aRMS tumors bear a signature chromosomal translocation resulting in the expression of the PAX3-FOXO1 fusion gene, which reactivates pro-proliferative embryonic skeletal muscle signaling pathways. Children with metastatic, PAX3-FOXO1-positive aRMS have the worst outcome, with a 5-year survival of <10%. High risk patients with PAX3-FOXO1-positive aRMS have few therapeutic options; however, the goal of our laboratory is to identify new drug targets tailored to the genetic changes associated with the disease.

Genetic modeling of aRMS

A genetically engineered mouse model (GEMM) of aRMS has elegantly shown a role for PAX3-FOXO1 in tumorigenesis.² Our laboratory has taken the complementary approach of modeling aRMS from primary (non-malignant) human skeletal muscle myoblasts (HSMMs). This is accomplished by the sequential expression in HSMMs of PAX3-FOXO1, followed by the catalytic subunit of telomerase (hTERT), followed by the MycN oncogene (model termed HSMM^PF+H+M). This approach transforms HSMMs in vitro and produces xenografts in vivo that histologically mimic human aRMS.³ Our model allows us to study aRMS initiation and step-wise progression in human cells, and to perform large-scale screens of new aRMS therapeutic targets in vivo.

Using our model, we have discovered that the evolutionarily conserved Hippo tumor suppressor pathway appears silenced in aRMS.This pathway is tightly regulated by PAX3-FOXO1-mediated regulation of Hippo pathway members. We propose that reactivation of the Hippo pathway will block tumorigenesis and provide a novel therapeutic strategy for aRMS.

Previous Studies

Identification of PAX3-FOXO1 Targets

We have shown that early introduction of PAX3-FOXO1 contributes to HSMM bypass of cellular senescence, and also primes cells for aRMS tumorigenesis.⁴ We thus hypothesized that transcriptional changes promoted by early PAX3-FOXO1 expression underlie this phenomenon. To examine the transcriptional changes regulated by PAX3-FOXO1, we examined primary HSMMs expressing PAX3-FOXO1 using microarray analysis. When compared to control myoblasts, the resulting ~2600 significantly changed genes included PAX3-FOXO1 targets that our laboratory and others have previously described, but also many novel targets. Upon further examination, we found that genes predicted to inhibit the Hippo pathway were elevated in PAX3-FOXO1-positive human aRMS cells and tumors, with elevated levels associated with worse clinical outcome. These data suggested to us that we had identified Hippo pathway inhibition as a vital component of aRMS biology that had not been described previously.

The Hippo Pathway and Cancer

The Hippo tumor suppressor pathway was so named for seminal studies performed in Drosophila melanogaster (fruit fly), where mutations in the hippo gene led to profound overgrowth phenotypes. Indeed, our understanding of this pathway is best in this model system. However, the Hippo signaling cascade is highly conserved, and Hippo pathway orthologs can be traced from single-celled organisms to humans.^5,6 In multi-cellular organisms, the Hippo pathway triggers growth arrest when tissues and organs have reached the appropriate size. Not surprisingly, cancers have evolved to silence the Hippo pathway, leading to increased proliferation and tumorigenesis. In mammals, the Hippo signaling pathway consists of a phospho-relay cascade whose core components are the MST1/2 kinases, Lats1/2 kinases, and the YAP/TAZ transcriptional activators. In Drosophila and mammals, disruption of this signaling pathway leads to tissue overgrowth and tumorigenic phenotypes.^7-17 In the case of soft tissue sarcomas, homozygous loss of Lats1 in mice leads to spontaneous soft tissue sarcomas in 14% of female mice and 83% of Lats-/- mice develop soft tissue sarcomas in response to specific carcinogenic treatments.¹⁸ In human soft tissue sarcomas, epigenetic downregulation has been observed for MST1/2 and Lats1 in primary tumors.^19,20

Although mechanisms of inhibition of the Hippo pathway have been described for other cancers (for example, methylation of the MST1 gene promoter or upregulation of YAP,^17,20,21 it appears that PAX3-FOXO1 evolved a novel way to block the Hippo tumor suppressor pathway in aRMS: by up-regulating endogenous inhibitors of the Hippo pathway.

Purpose of Investigation

We have discovered that the inhibition of the Hippo pathway is a vital signaling node for aRMS. We hypothesize that inactivation of the Hippo pathway is necessary for aRMS initiation and maintenance, while reactivation of the Hippo pathway may be a new aRMS therapeutic strategy. The studies below will begin to define the biology of Hippo signaling in aRMS, and identify points for experimental and therapeutic exploitation.

Research Plan and Experimental Design

1. Determine how the Hippo pathway is inhibited in aRMS.

The mechanism for regulation of the Hippo pathway in aRMS is unknown, but we hypothesize that an inhibitory protein complex regulated by PAX3-FOXO1 target genes is responsible. We propose to identify the members of this complex so that we can better understand the mechanism of Hippo pathway inhibition, and begin to identify proteins amenable to pharmacologic manipulation for therapeutics.

Proteomic Identification of Hippo Pathway Inhibitors

To achieve this task, we will use an unbiased proteomics approach utilizing immunoprecipitation (IP) of epitope (FLAG)-tagged PAX3-FOXO1 target genes. These genes will be stably expressed in Rh28 or HSMM^PF+H+M aRMS cells, and will be immunopurified from cell lysates using anti-FLAG antibodies. Associated proteins will be eluted using FLAG peptide. Lysates from cells expressing an empty vector will identify non-specific proteins that bind to anti-FLAG or resin used for IPs. Associated proteins will be subject to in-solution tryptic digestion and identified by tandem mass spectrometry (LC-MS/MS).

As we could potentially capture dozens of proteins, interactors will be prioritized using pathway mapping engines, including Ingenuity Pathway Analysis. Then, a more restrictive list will be generated based on significance in our biologic system and "druggability." Interactors will be further examined in vitro for (i) direct versus indirect association with proteins in the Hippo pathway (using in vitro translated proteins to test direct protein-protein interaction), (ii) ability to inhibit the Hippo pathway performing experiments in HEK293T cells,²² and (iii) the domain required for protein-protein interactions (using deletion and/or NAAIRS mutagenesis.²³ Last (iv), interactors will be tested for their ability to support tumorigenesis in vivo. This will be accomplished using the doxycyline (dox)-inducible shRNA system (Figure 5). Briefly, for each interactor five shRNAs will be designed and tested for knockdown in vitro. Then, the three causing most knockdown will be subcloned into a dox-inducible lentiviral backbone. aRMS cells expressing inducible plasmids will injected as xenografts into SCID/ beige mice. When tumors are palpable, mice will be given water supplemented with 1mg/mL dox to induce shRNA expression, and monitored twice weekly to measure tumor growth. After 30 days of dox treatment or when animals meet stopping rules, animals will be sacrificed and necropsied, with harvested tumors analyzed by immunohistochemistry to measure proliferation (Ki67), apoptosis (TUNEL), and angiogenesis (CD33),²⁴ and also to prove target knockdown by the expressed shRNA. This unbiased approach will identify interactors of the Hippo signaling pathway and characterize their role in inhibiting the Hippo pathway in aRMS.

PP2A as a candidate for inhibition of the Hippo pathway.

To complement the unbiased approach above, we will also use a candidate approach. In Drosophila, inhibitory Hippo signaling complexes contain the PP2A phosphatase, which inhibits the MST1 kinase.²⁹ Although the precise target of PP2A in this complex is not known, it has been shown that inhibition of PP2A synergizes with other agents to inhibit tumorigenesis. To determine if PP2A inhibition can reactivate MST1 signaling in aRMS, we will treat aRMS Rh28 cells with PP2A small molecule inhibitors in vitro, and assess the subsequent effect on PP2A activity,³⁰ MST1 kinase activity, and cell proliferation.

If the above in vitro studies support a role for PP2A inhibition in aRMS treatment, we will evaluate PP2A inhibitors in our aRMS xenograft system described above. We predict that PP2A inhibition will block aRMS cell proliferation and tumor growth, but for validation will examine harvested tumors by IHC for phosphorylated MST1 to prove that the Hippo pathway is reactivated.

2. Examine the requirement for YAP/TAZ in aRMS tumorigenesis in vivo.

We have shown that the driving mutation in aRMS, PAX3-FOXO1, manipulates the Hippo pathway by upregulating endogenous Hippo pathway inhibitors. However, whether YAP or TAZ (as the most distal members of the Hippo pathway) mediates aRMS tumorigenesis is unknown. YAP has been studied more extensively than TAZ in cancer, therefore we will begin by examining the function of YAP in aRMS. To address this, we will use aRMS murine xenografts to evaluate the impact of genetic and pharmacologic YAP loss-of-function on aRMS tumor maintenance, and substitution studies in our genetic model of aRMS to evaluate whether YAP can functionally replace PAX3-FOXO1 in tumor initiation.

Is YAP necessary for aRMS tumor maintenance in vivo?

First, we will stably express dox-inducible YAP shRNAs (or control vectors) in Rh28 and HSMM^PF+H+M cells, then test them as xenografts as described above. We predict that loss of YAP expression by induction of shRNAs will block proliferation and subsequent xenograft tumor growth of both Rh28 cells and HSMM^PF+H+M cells.

Next, we will use a pharmacologic approach to block Yap signaling. Very recently, the commercially available compound verteporfin (Novartis) was found to inhibit Yap-mediated proliferation in vitro and in vivo.³¹ Verteporfin is approved for the treatment of macular degeneration in humans. We will test verteporfin in both Rh28 and HSMM^PF+H+M xenografts. As above, when xenografts become palpable, verteporfin (or DMSO vehicle) will be administered by intraperitoneal injection every other day.³¹ We predict that inhibiting YAP through verteporfin will block tumor growth. If successful, these experiments will provide preclinical evidence for evaluation of verteporfin in patients with aRMS.

Is YAP sufficient for aRMS initiation in vivo?

The power of our genetic model of aRMS is the ability to perform substitution experiments to identify genes that regulate aRMS initiation. In this way, we will examine whether the YAP oncogene can replace PAX3-FOXO1 in modeling aRMS. We will use primary HSMM cells transduced with YAP, hTERT, and MycN (in that order) to generate HSMM^Y+H+M cell lines to compare with HSMM^M+H (negative control), and HSMM^PF+H+M (positive control) cell lines. These will be tested as xenografts as described above. We predict that YAP will substitute for PAX3-FOXO1, and HSMM^Y+H+M cells will be capable of forming tumors in mice.

Impact Statement

Survival for children with aRMS has not improved appreciably in over 30 years. While PAX3-FOXO1 is an attractive therapeutic target, current treatments are not capable of blocking its oncogenic activity. Instead, efforts are aimed at identifying cooperating proteins and signaling pathways. We have found that PAX3-FOXO1 upregulates endogenous Hippo pathway inhibitors, resulting in Hippo pathway suppression, promotion of cellular proliferation, and ultimately tumorigenesis. Our findings highlight a Hippo-suppressing role for PAX3-FOXO1, and implicate the Hippo pathway in aRMS. As this pathway is highly conserved, we predict that Hippo pathway dysregulation will be manifest in other pediatric cancers. Indeed, the Yap oncogene was recently implicated in Ewing's sarcoma.³² Therefore, our studies will have direct impact for aRMS, and we hope to expand our findings to find new therapeutic strategies for other pediatric sarcomas.

References

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4. Linardic CM, Naini S, Herndon JE, 2nd, Kesserwan C, Qualman SJ, Counter CM. The PAX3-FKHR fusion gene of rhabdomyosarcoma cooperates with loss of p16INK4A to promote bypass of cellular senescence. Cancer Res. 2007 Jul 15;67(14):6691-9.

5. Hilman D, Gat U. The evolutionary history of YAP and the hippo/YAP pathway. Mol Biol Evol. 2011 Aug;28(8):2403-17.

6. Sebe-Pedros A, Zheng Y, Ruiz-Trillo I, Pan D. Premetazoan origin of the hippo signaling pathway. Cell Rep. 2012 Jan 26;1(1):13-20.

7. Pan D. The hippo signaling pathway in development and cancer. Dev Cell. 2010 Oct 19;19(4):491-505.

8. Bao Y, Hata Y, Ikeda M, Withanage K. Mammalian Hippo pathway: from development to cancer and beyond. J Biochem. 2011 Apr;149(4):361-79.

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10. Hall CA, Wang R, Miao J, Oliva E, Shen X, Wheeler T, et al. Hippo pathway effector Yap is an ovarian cancer oncogene. Cancer Res. 2010 Nov 1;70(21):8517-25.

11. Lai D, Ho KC, Hao Y, Yang X. Taxol resistance in breast cancer cells is mediated by the hippo pathway component TAZ and its downstream transcriptional targets Cyr61 and CTGF. Cancer Res. 2011 Apr 1;71(7):2728-38.

12. Liu AM, Xu MZ, Chen J, Poon RT, Luk JM. Targeting YAP and Hippo signaling pathway in liver cancer. Expert Opin Ther Targets. 2010 Aug;14(8):855-68.

13. Visser-Grieve S, Hao Y, Yang X. Human homolog of Drosophila expanded, hEx, functions as a putative tumor suppressor in human cancer cell lines independently of the Hippo pathway. Oncogene. 2011 Jul 25.

14. Zeng Q, Hong W. The emerging role of the hippo pathway in cell contact inhibition, organ size control, and cancer development in mammals. Cancer Cell. 2008 Mar;13(3):188-92.

15. Zhang X, George J, Deb S, Degoutin JL, Takano EA, Fox SB, et al. The Hippo pathway transcriptional co-activator, YAP, is an ovarian cancer oncogene. Oncogene. 2011 Jun 23;30(25):2810-22.

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Grant Funding

The Liddy Shriver Sarcoma Initiative announced the funding of this $50,000 grant in August 2012. This grant was made possible by generous gifts from the Jordan Paganelli Sarcoma Foundation and JRock-n-Run (in memory of Jordan Scott Paganelli), and from the families and friends of Timothy "Tim" Yeates, Anna Rogotzke, Dillon Wolford, Ashley Miller, Harper Creek and Teri Marriage-Kuespert, all of whom lost their lives to rhabdomyosarcoma.

Duke University published a news story about this study.

A characteristic genetic change found in many cases of aRMS is the PAX3-FOXO1 fusion gene. This occurs through a chromosomal translocation between two transcription factor genes: the PAX3 gene on Chromosome 2 and the FOXO1 gene (also known as FKHR) on Chromosome 13. The breakpoints on these two chromosomes are midway between the coding regions of these genes, resulting in a fusion gene that contains the DNA binding region of PAX3 and the transactivation domain of FOXO1. Dysregulated gene expression caused by the PAX3-FOXO1 fusion gene is believed to be responsible for aRMS tumorigenic properties.
Generally, xenografts are the transplant of cells or tissues from one organism into another. For aRMS xenografts, we use human aRMS cells transplanted into mice. First, aRMS cells are grown in vitro. Cells are then harvested and prepared for implantation in the mouse. To prevent rejection of the human cells, we use immune-compromised mice. Our laboratory uses the SCID/beige strain, which is deficient in T and B lymphocytes, and contains defects in T cells and macrophages. After implantation of the aRMS cells, within 2-3 weeks tumors begin to grow. These tumors can be isolated and examined for tumorigenic markers by immunohistochemistry.
The Hippo pathway is a phospho-relay cascade that regulates the YAP/TAZ oncogenes. The core members of the Hippo pathway in mammals are the MST1/2 protein kinases, which phosphorylate and activate downstream Lats1/2 protein kinases, which phosphorylate YAP/TAZ. When phosphorylated, YAP/TAZ are believed to be inactive, and sequestered in the cytoplasm. Activation of this cascade serves a tumor suppressive role. When YAP/TAZ are unphosphorylated, they can translocate to the nucleus and serve as oncogenes by facilitating upregulation of pro-proliferative genes.
Immunoprecipitation is a method to isolate specific proteins from a protein mixture. Cultured cells are broken open by cell lysis and a proteins mixture is isolated. Antibodies specific to a protein of interest are added to the protein mixture. This antibody will bind the protein of interest. Protein G-conjugated agarose beads are then added, which will bind the antibody. These beads, bound with antibody and the protein of interest, can then be washed to remove the remaining protein mixture. Often, the immunopurified protein is associated with other proteins, which help it perform its biological function. These so-called "co-precipitated" proteins are what we hope to identify through immunoprecipitation and mass spectrometry identification as described.
One method of examining a gene’s function is to block its expression through RNA interference, or RNAi. For detailed information on RNAi, the reader is directed to one of the many excellent reviews of this process.^25-27 To induce RNAi experimentally, one method that is routinely used is the expression of short hairpin RNAs, or shRNAs. Our laboratory uses an inducible shRNA expression system.²⁸ Doxycycline (dox) treatment is used to induce expression of specific shRNAs. In this system, cells express plasmids containing a Tet-repressor element (TRE) and the Tet-repressor (TetR). In the absence of dox, the TetR binds the TRE, blocking shRNA expression. In the presence of dox, dox binds and sequesters the TetR, allowing shRNA expression. These shRNAs can then perform gene suppression by the RNAi pathway.
RASSF4 expression in RMS cell lines (A) and human tumors (B) shows correlation between PAX3-FOXO1 and RASSF4 expression. C) RASSF4 expression is associated with worse clinical prognosis. © ASCI, image from (1).
RASSF4 expression in RMS cell lines (A) and human tumors (B) shows correlation between PAX3-FOXO1 and RASSF4 expression. C) RASSF4 expression is associated with worse clinical prognosis. © ASCI, image from (1).
RASSF4 expression in RMS cell lines (A) and human tumors (B) shows correlation between PAX3-FOXO1 and RASSF4 expression. C) RASSF4 expression is associated with worse clinical prognosis. © ASCI, image from (1).
A) Lentiviral RASSF4 shRNA plasmids were validated in HEK293T cells expressing HA-RASSF4 and measured by immunoblot. Actin immunoblot was used as a loading control. Loss of RASSF4 expression in HSMMPF+H+M cells leads to deficient cell proliferation (B), induction of senescence associated β-Gal staining (C), loss of BrdU incorporation (D), and induction of p21 protein (E) *p < 0.0001. © ASCI, image from (1).
A) Doxycycline (Dox) – inducible RASSF4 shRNA recapitulates constitutive RASSF4 shRNA. B) Loss of RASSF4 in aRMS xenografts leads to decreased tumor progression. © ASCI, image from (1).
A) Domain architecture of HA-RASSF4 constructs. B) RASSF4 associates with MST1 in aRMS cells in a RASSF4 SARAH domain-dependent fashion. Anti-HA immunoprecipitates from HSMMPF+H+M cells expressing HA-RASSF4, HA-RASSF4SARAH, or vector were used to examine association with MST1 or Pan Ras by immunoblot. These results were confirmed by immunopurifying endogenous MST1, and blotting for HA-RASSF4 or HA-RASSF4SARAH (C). © ASCI, image from (1).
RASSF4 loss leads to MST1 activation, as measured by P-Mob1 immunoblot. This effect was completely reversed by expression of MST1 K59R. Note the reversal of p21 induction in cells expressing MST1 K59R and RASSF4 shRNA. © ASCI, image from (1).
A) Kinase-dead MST1 K59R prevents senescence induction caused by RASSF4 loss. HSMMPF+H+M cells stably expressing vector or MST1 K59R were transduced with vector or RASSF4 shRNAs. Senescence induction was measured by B-gal assay. *p < 0.00005, #p < 0.005. B) MST1 K59R partially blocks G0/G1 accumulation in RASSF4-deficient cells, as measured by cell cycle analysis. © ASCI, image from (1).
Left: Representative images from RMS tissue microarrays immunostained for YAP protein. Scale bars=100µm. Right: quantitation of YAP immunostained RMS tissue microarrays. Muscle N=11, eRMS N=58, aRMS N=72. Error bars represent SEM. *p<0.0001, Mann-Whitney test. © ASCI, image from (1).
YAP loss-of-function (A) leads to growth arrest and senescence as measured by BrdU incorporation (B) and -gal staining (C). *p < 0.0001. © ASCI, image from (1).