Distinctive Clinicopathologic Features of Monomorphic B-cell Post-transplant Lymphoproliferative Disorders in Children.

INTRODUCTION: Post-transplant lymphoproliferative disorders (PTLDs) comprise a heterogeneous group of Epstein-Barr virus (EBV)-positive or negative lymphoid or plasmacytic lesions in solid organ or hematopoietic stem cell (HSC) transplant recipients. Although PTLDs in adults have been extensively studied, the clinicopathologic features of monomorphic B-cell PTLD in children, particularly EBV-negative forms, are still poorly understood. METHODS: We retrospectively reviewed all our pediatric cases of monomorphic B-cell PTLDs diagnosed in the past 10 years. Clinical data were reviewed. Pathologic data including histologic types and EBV status were analyzed. Additional immunohistochemical stains, FISH studies, and TP53 gene mutational status were performed. RESULTS: 4 of 18 cases were EBV-negative. All 4 EBV-negative cases were strikingly confined to the gastrointestinal (GI) tract or abdominal lymph nodes, while tumors in EBV-positive cases were found at various anatomic sites; 2 of 4 EBV-negative cases carried mutations in TP53 gene. Our cohort also included 2 rare types of PTLD, one plasmablastic lymphoma and one high-grade B-cell lymphoma, not otherwise specified (HGBL, NOS). CONCLUSION: We report that monomorphic B-cell PTLDs in children have distinctive clinical and pathological features. More studies are needed to clarify whether and how much these pediatric PTLDs differ from their adult counterparts.

Transcript analysis for variant classification resolution in a child with primary ciliary dyskinesia.

Transcriptional analysis can be utilized to reconcile variants of uncertain significance, particularly those predicted to impact splicing. Laboratory analysis of the predicted mRNA transcript may allow inference of the in vivo impact of the variant and aid prediction of its clinical significance. We present a patient with classical features of primary ciliary dyskinesia (PCD) who was identified to have compound heterozygous variants in the DNAH11 gene (c.10691 + 2T > C, c.13523_13543dup21) via trio whole-exome sequencing in 2013. These variants were originally classified as Mutation and Likely Mutation. However, these variants were downgraded to variants of uncertain significance (VUSs) during reanalysis in 2016 because of uncertainty that they caused a loss of function of the gene. c.10691 + 2T > C is predicted to abrogate the canonical splice site and lead to the skipping of exon 65, but the adjoining of exon 64 and exon 66 in the DNAH11 transcript preserves the reading frame of the resultant protein. c.13523_13543dup21 is located in the last exon of the DNAH11 coding sequence, upstream of the canonical stop codon, which suggests a reduced likelihood to trigger nonsense-mediated decay (NMD). Transcriptional analysis was performed to characterize the impact of the variants, resulting in reclassification of c.10691 + 2T > C to Likely Pathogenic by providing evidence that it results in a deleterious effect and subsequent downstream reclassification of c.13523_13543dup21 to Likely Pathogenic as well. Our case illustrates the potential impact of transcriptional analysis on variant resolution, supporting its usage on variants that exert an unpredictable effect on splicing.

A case of aberrant CD8 T cell-restricted IL-7 signaling with a Janus kinase 3 defect-associated atypical severe combined immunodeficiency.

Severe combined immunodeficiency (SCID) disorders compromise lymphocyte numbers and/or function. One subset of SCID typically affects T cell and Natural Killer (NK) cell development in tandem (T-B+NK-) due to mutations arising in the genes encoding the common γ chain or Janus Kinase 3 (JAK3). In rare circumstances, mutations in the JAK3 gene have been reported to cause atypical SCID that selectively affects T cells (T-B+NK+). Here we describe a case involving a female infant who was referred to our institution on day nine of life following an abnormal newborn screen result for T-SCID. Immunological assessments revealed a T-B+NK+ phenotype and molecular analyses, including whole exome sequencing, identified compound heterozygous JAK3 variants (R117C and E658K). Pre-transplant phosflow analyses revealed a persistent IL-7 signaling defect, based on phospho-STAT5 measurements, only in CD8 but not CD4 T cells. Intriguingly, phospho-STAT5 signals in response to IL-2 stimulation were not affected in either CD4 or CD8 T cells. The pre-transplant clinical course was unremarkable, and the patient received a cord-blood stem cell transplant on day 716 of life. Post-transplant monitoring revealed that despite normalization of lymphocyte counts, the CD8 T cell-restricted IL-7 signaling defect was still evident at day 627 post-transplant (phospho-STAT5 signal in CD8 T cells was > 60% reduced compared with CD4 T cells). The post-transplant clinical course has also been complicated by identification of autoimmune responses and likely GVHD-induced ichthyosis. To the best of our knowledge, this report represents the third case of JAK3-associated atypical SCID reported in the literature.

The classification of pediatric and young adult renal cell carcinomas registered on the children's oncology group (COG) protocol AREN03B2 after focused genetic testing.

BACKGROUND: Renal cell carcinomas (RCCs) are rare in young patients. Knowledge of their pathologic and molecular spectrum remains limited, and no prospective studies have been performed to date in this population. This study analyzes patients diagnosed with RCC who were prospectively enrolled in the AREN03B2 Children's Oncology Group (COG). The objective was to classify these tumors with the aid of focused genetic testing and to characterize their features. METHODS: All tumors registered as RCC by central review were retrospectively re-reviewed and underwent additional ancillary studies. Tumors were classified according to the 2016 World Health Organization classification system when possible. RESULTS: In total, 212 tumors were identified, and these were classified as microphthalmia transcription factor (MiT) translocation RCC (MiT-RCC) (41.5%), papillary RCC (16.5%), renal medullary carcinoma (12.3%), chromophobe RCC (6.6%), clear cell RCC (3.3%), fumarate hydratase-deficient RCC (1.4%), and succinate dehydrogenase-deficient RCC (0.5%). Other subtypes included tuberous sclerosis-associated RCC (4.2%), anaplastic lymphoma kinase (ALK)-rearranged RCC (3.8%), thyroid-like RCC (1.4%), myoepithelial carcinoma (0.9%), and unclassified (7.5%). MiT-RCCs were classified as either transcription factor E3 (TFE3) (93.2%) or EB (TFEB) (6.8%) translocations, and characterization of fusion partners was possible in most tumors. CONCLUSIONS: The current study delineates the frequency of distinct RCC subtypes in a large prospective series of young patients and contributes knowledge to the diagnostic, clinical, and genetic features of MiT-RCC, the most common subtype among this age group. The identification of rare subtypes expands the spectrum of RCC in young patients, supporting the need for a thorough diagnostic workup. These studies may aid in the introduction of specific therapies for different RCC subtypes in the future. Cancer 2018. © 2018 American Cancer Society.

Genetic Testing Requires NGS and Sanger Methodologies.

Investigators from the EuroEPINOMICS rare epilepsy syndromes Dravet working group performed whole-exome sequencing on 31 trios that had been reported negative for SCN1A mutations by Sanger sequencing.

Divergence(s) in nodal signaling between aggressive melanoma and embryonic stem cells.

The significant role of the embryonic morphogen Nodal in maintaining the pluripotency of embryonic stem cells is well documented. Interestingly, the recent discovery of Nodal's re-expression in several aggressive and metastatic cancers has highlighted its critical role in self renewal and maintenance of the stem cell-like characteristics of tumor cells, such as melanoma. However, the key TGFß/Nodal signaling component(s) governing Nodal's effects in metastatic melanoma remain mostly unknown. By employing receptor profiling at the mRNA and protein level(s), we made the novel discovery that embryonic stem cells and metastatic melanoma cells share a similar repertoire of Type I serine/threonine kinase receptors, but diverge in their Type II receptor expression. Ligand:receptor crosslinking and native gel binding assays indicate that metastatic melanoma cells employ the heterodimeric TGFß receptor I/TGFß receptor II (TGFßRI/TGFßRII) for signal transduction, whereas embryonic stem cells use the Activin receptors I and II (ACTRI/ACTRII). This unexpected receptor usage by tumor cells was tested by: neutralizing antibody to block its function; and transfecting the dominant negative receptor to compete with the endogenous receptor for ligand binding. Furthermore, a direct biological role for TGFßRII was found to underlie vasculogenic mimicry (VM), an endothelial phenotype contributing to vascular perfusion and associated with the functional plasticity of aggressive melanoma. Collectively, these findings reveal the divergence in Nodal signaling between embryonic stem cells and metastatic melanoma that can impact new therapeutic strategies targeting the re-emergence of embryonic pathways.

Nodal signaling promotes a tumorigenic phenotype in human breast cancer.

The Ras-ERK pathway is deregulated in approximately a third of human cancers, particularly those of epithelial origin. In aggressive, triple-negative, basal-like breast cancers, most tumors display increased MEK and ERK phosphorylation and exhibit a gene expression profile characteristic of Kras or EGFR mutant tumors; however, Ras family genetic mutations are uncommon in triple-negative breast cancer and EGFR mutations account for only a subset of these tumors. Therefore, the upstream events that activate MAPK signaling and promote tumor aggression in triple-negative breast cancers remain poorly defined. We have previously shown that a secreted TGF-ß family signaling ligand, Nodal, is expressed in breast cancer in correlation with disease progression. Here we highlight key findings demonstrating that Nodal is required in aggressive human breast cancer cells to activate ERK signaling and downstream tumorigenic phenotypes both in vitro and in vivo. Experimental knockdown of Nodal signaling downregulates ERK activity, resulting in loss of c-myc, upregulation of p27, G1 cell cycle arrest, increased apoptosis and decreased tumorigenicity. The data suggest that ERK activation by Nodal signaling regulates c-myc and p27 proteins post-translationally and that this cascade is essential for aggressive breast tumor behavior in vivo. As the MAPK pathway is an important target for treating triple-negative breast cancers, upstream Nodal signaling may represent a promising target for breast cancer diagnosis and combined therapies aimed at blocking ERK pathway activation.

Tumor cell vasculogenic mimicry: from controversy to therapeutic promise.

In 1999, The American Journal of Pathology published an article entitled "Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry," by Maniotis and colleagues, which ignited a spirited debate for several years and earned distinction as a citation classic. Tumor cell vasculogenic mimicry (VM) refers to the plasticity of aggressive cancer cells forming de novo vascular networks, which thereby contribute to perfusion of rapidly growing tumors, transporting fluid from leaky vessels, and/or connecting with the constitutional endothelial-lined vasculature. The tumor cells capable of VM share a plastic, transendothelial phenotype, which may be induced by hypoxia. Since VM was introduced as a novel paradigm for melanoma tumor perfusion, many studies have contributed new findings illuminating the underlying molecular pathways supporting VM in a variety of tumors, including carcinomas, sarcomas, glioblastomas, astrocytomas, and melanomas. Facilitating the functional plasticity of tumor cell VM are key proteins associated with vascular, stem cell, and hypoxia-related signaling pathways, each deserving serious consideration as potential therapeutic targets and diagnostic indicators of the aggressive, metastatic phenotype.

Molecular pathways: vasculogenic mimicry in tumor cells: diagnostic and therapeutic implications.

Tumor cell vasculogenic mimicry (VM) describes the functional plasticity of aggressive cancer cells forming de novo vascular networks, thereby providing a perfusion pathway for rapidly growing tumors, transporting fluid from leaky vessels, and/or connecting with endothelial-lined vasculature. The underlying induction of VM seems to be related to hypoxia, which may also promote the plastic, transendothelial phenotype of tumor cells capable of VM. Since its introduction in 1999 as a novel paradigm for melanoma tumor perfusion, many studies have contributed new insights into the underlying molecular pathways supporting VM in a variety of tumors, including melanoma, glioblastoma, carcinomas, and sarcomas. In particular, critical VM-modulating genes are associated with vascular (VE-cadherin, EphA2, VEGF receptor 1), embryonic and/or stem cell (Nodal, Notch4), and hypoxia-related (hypoxia-inducible factor, Twist1) signaling pathways. Each of these pathways warrants serious scrutiny as potential therapeutic, vascular targets, and diagnostic indicators of plasticity, drug resistance, and the aggressive metastatic phenotype.

Nodal expression and detection in cancer: experience and challenges.

Nodal is a TGF-ß-related embryonic morphogen that is expressed in multiple human cancers. Detection of Nodal expression in these tissues can be challenging if issues related to Nodal transcription and protein processing are not considered. Here, we discuss certain characteristics related to Nodal expression and function and how these can facilitate acquisition and interpretation of expression data, contributing to our understanding of the potential role of Nodal in human cancer. We also discuss how Nodal could be exploited clinically as a novel biomarker for cancer progression and therapeutic target.

Lysyl oxidase contributes to mechanotransduction-mediated regulation of transforming growth factor-ß signaling in breast cancer cells.

Transforming growth factor-ß (TGF-ß) regulates all stages of mammary gland development, including the maintenance of tissue homeostasis and the suppression of tumorigenesis in mammary epithelial cells (MECs). Interestingly, mammary tumorigenesis converts TGF-ß from a tumor suppressor to a tumor promoter through molecular mechanisms that remain incompletely understood. Changes in integrin signaling and tissue compliance promote the acquisition of malignant phenotypes in MECs in part through the activity of lysyl oxidase (LOX), which regulates desmoplastic reactions and metastasis. TGF-ß also regulates the activities of tumor reactive stroma and MEC metastasis. We show here that TGF-ß1 stimulated the synthesis and secretion of LOX from normal and malignant MECs in vitro and in mammary tumors produced in mice. The ability of TGF-ß1 to activate Smad2/3 was unaffected by LOX inactivation in normal MECs, whereas the stimulation of p38 MAPK by TGF-ß1 was blunted by inhibiting LOX activity in malignant MECs or by inducing the degradation of hydrogen peroxide in both cell types. Inactivating LOX activity impaired TGF-ß1-mediated epithelial-mesenchymal transition and invasion in breast cancer cells. We further show that increasing extracellular matrix rigidity by the addition of type I collagen to three-dimensional organotypic cultures promoted the proliferation of malignant MECs, a cellular reaction that was abrogated by inhibiting the activities of TGF-ß1 or LOX, and by degrading hydrogen peroxide. Our findings identify LOX as a potential mediator that couples mechanotransduction to oncogenic signaling by TGF-ß1 and suggest that measures capable of inactivating LOX function may prove effective in diminishing breast cancer progression stimulated by TGF-ß1.

Regulation of the embryonic morphogen Nodal by Notch4 facilitates manifestation of the aggressive melanoma phenotype.

Metastatic melanoma is an aggressive skin cancer associated with poor prognosis. The reactivation of the embryonic morphogen Nodal in metastatic melanoma has previously been shown to regulate the aggressive behavior of these tumor cells. During the establishment of left-right asymmetry in early vertebrate development, Nodal expression is specifically regulated by a Notch signaling pathway. We hypothesize that a similar relationship between Notch and Nodal may be reestablished in melanoma. In this study, we investigate whether cross talk between the Notch and Nodal pathways can explain the reactivation of Nodal in aggressive metastatic melanoma cells. We show a molecular link between Notch and Nodal signaling in the aggressive melanoma cell line MV3 via the activity of an RBPJ-dependent Nodal enhancer element. We show a precise correlation between Notch4 and Nodal expression in multiple aggressive cell lines but not poorly aggressive cell lines. Surprisingly, Notch4 is specifically required for expression of Nodal in aggressive cells and plays a vital role both in the balance of cell growth and in the regulation of the aggressive phenotype. In addition, Notch4 function in vasculogenic mimicry and anchorage-independent growth in vitro is due in part to Notch4 regulation of Nodal. This study identifies an important role for cross talk between Notch4 and Nodal in metastatic melanoma, placing Notch4 upstream of Nodal, and offers a potential molecular target for melanoma therapy.

New insights into cathepsin D in mammary tissue development and remodeling.

Cathepsin D (CatD) is a lysosomal aspartyl endopeptidase originally considered a "house keeping enzyme" involved in the clearance of unwanted proteins. However, recent studies have revealed CatD's involvement in apoptosis and autophagy, thus signifying an important function in the proper development and maintenance of multi-cellular organs. In the mammary gland, matrix degradation and the remodeling process are orchestrated by proteolytic enzymes, but the role of CatD at distinct developmental stages has remained mostly unexplored. Based on our previous studies we sought to address the role of this endopeptidase in mammary gland development and remodeling. By employing a mouse model, we report a previously unidentified participation of CatD in different stages of mammary gland development. Our findings reveal that CatD undergoes distinct protein processing at different stages of mammary gland development, and this customized processing results in differential enzymatic activity (constitutive and low pH activatable) best fitting particular stage(s) of development. In addition, at the onset of involution the N-glycan structure of this endopeptidase switches from a mixed high mannose and hybrid structure to an almost exclusively high mannose type, but reverts back to the original N-glycan composition by day 4 of involution. Our findings illuminate (at least in part) the "raison d'être" for CatD's numerous and highly regulated proteolytic processing steps from the pro-form to the mature enzyme. In the mammary gland, specific cleavage product(s) perform specialized function(s) befitting each stage of remodeling. It is noteworthy that deregulated synthesis, secretion and glycosylation of CatD are hallmarks of cancer progression. Thus, identifying the role of CatD in a dynamic normal tissue undergoing highly regulated cycles of remodeling could provide valuable information illuminating the deregulation of CatD associated with cancer development and metastasis.

Development and cancer: at the crossroads of Nodal and Notch signaling.

Aggressive tumor cells express a plastic, multipotent phenotype similar to embryonic stem cells. However, the absence of major regulatory checkpoints in these tumor cells allows aberrant activation of embryonic signaling pathways, which seems to contribute to their plastic phenotype. Emerging evidence showing the molecular cross-talk between two major stem cell signaling pathways Nodal and Notch suggests a promising therapeutic strategy that could target aggressive tumor cells on the basis of their unique plasticity, and provide new insights into the mechanisms underlying the re-emergence of developmental signaling pathways during tumor progression.

Epigenetically reprogramming metastatic tumor cells with an embryonic microenvironment.

UNLABELLED: We have previously shown that the microenvironment of human embryonic stem cells (hESCs) is able to change and reprogram aggressive cancer cells to a less aggressive state. Some mechanisms implicated in the phenotypic changes observed after this exposure are mainly associated with the Nodal signaling pathway, which plays a key role in tumor cell plasticity. However, several other molecular mechanisms might be related directly and/or indirectly to these changes, including microRNA (miRNA) regulation and DNA methylation. AIM: To further explore the epigenetic mechanisms potentially underlying the phenotypic changes that occur after exposing metastatic melanoma cells to a hESC microenvironment. MATERIALS & METHODS: A total of 365 miRNAs were screened using the TaqMan® Low Density Arrays. We also evaluated whether DNA methylation could be one of the factors regulating the expression of the inhibitor of Nodal, Lefty, in hESCs (where it is highly expressed) vs melanoma cells (where it is not expressed). RESULTS: Using these experimental approaches, we identified miRNAs that are up- and down-regulated in melanoma cells exposed to a hESC microenvironment, such as miR-302a and miR-27b, respectively. We also demonstrate that Notch4 is one of the targets of miR-302a, which is upstream of Nodal. Additionally, one of the mechanisms that might explain the absence of the inhibitor of Nodal, Lefty, in cancer cells is silencing by DNA methylation, which provides new insights into the unregulated expression of Nodal in melanoma. CONCLUSION: These findings suggest that epigenetic changes such as DNA methylation and regulation by microRNAs might play a significant role in tumor cell plasticity and the metastatic phenotype.

Human embryonic stem cell microenvironment suppresses the tumorigenic phenotype of aggressive cancer cells.

Embryonic stem cells sustain a microenvironment that facilitates a balance of self-renewal and differentiation. Aggressive cancer cells, expressing a multipotent, embryonic cell-like phenotype, engage in a dynamic reciprocity with a microenvironment that promotes plasticity and tumorigenicity. However, the cancer-associated milieu lacks the appropriate regulatory mechanisms to maintain a normal cellular phenotype. Previous work from our laboratory reported that aggressive melanoma and breast carcinoma express the embryonic morphogen Nodal, which is essential for human embryonic stem cell (hESC) pluripotency. Based on the aberrant expression of this embryonic plasticity gene by tumor cells, this current study tested whether these cells could respond to regulatory cues controlling the Nodal signaling pathway, which might be sequestered within the microenvironment of hESCs, resulting in the suppression of the tumorigenic phenotype. Specifically, we discovered that metastatic tumor cells do not express the inhibitor to Nodal, Lefty, allowing them to overexpress this embryonic morphogen in an unregulated manner. However, exposure of the tumor cells to a hESC microenvironment (containing Lefty) leads to a dramatic down-regulation in their Nodal expression concomitant with a reduction in clonogenicity and tumorigenesis accompanied by an increase in apoptosis. Furthermore, this ability to suppress the tumorigenic phenotype is directly associated with the secretion of Lefty, exclusive to hESCs, because it is not detected in other stem cell types, normal cell types, or trophoblasts. The tumor-suppressive effects of the hESC microenvironment, by neutralizing the expression of Nodal in aggressive tumor cells, provide previously unexplored therapeutic modalities for cancer treatment.

Hypoxia/reoxygenation: a dynamic regulator of lysyl oxidase-facilitated breast cancer migration.

Fluctuating oxygen levels characterize the microenvironment of many cancers and tumor hypoxia is associated with increased invasion and metastatic potential concomitant with a poor prognosis. Similarly, the expression of lysyl oxidase (LOX) in breast cancer facilitates tumor cell migration and is associated with estrogen receptor negative status and reduced patient survival. Here we demonstrate that hypoxia/reoxygenation drives poorly invasive breast cancer cells toward a more aggressive phenotype by up-regulating LOX expression and catalytic activity. Specifically, hypoxia markedly increased LOX protein expression; however, catalytic activity (beta-aminopropionitrile inhibitable hydrogen peroxide production) was significantly reduced under hypoxic conditions. Moreover, poorly invasive breast cancer cells displayed a marked increase in LOX-dependent FAK/Src activation and cell migration following hypoxia/reoxygenation, but not in response to hypoxia alone. Furthermore, LOX expression is only partially dependent on hypoxia inducible factor-1 (HIF-1alpha) in poorly invasive breast cancer cells, as hypoxia mimetics and overexpression of HIF-1alpha could not up-regulate LOX expression to the levels observed under hypoxia. Clinically, LOX expression positively correlates with tumor progression and co-localization with hypoxic regions (defined by HIF-1alpha expression) in ductal carcinoma in situ and invasive ductal carcinoma primary tumors. However, positive correlation is lost in metastatic tumors, suggesting that LOX expression is independent of a hypoxic environment at later stages of tumor progression. This work demonstrates that both hypoxia and reoxygenation are necessary for LOX catalytic activity which facilitates breast cancer cell migration through a hydrogen peroxide-mediated mechanism; thereby illuminating a potentially novel mechanism by which poorly invasive cancer cells can obtain metastatic competency.

Paradoxical roles for lysyl oxidases in cancer--a prospect.

Lysyl oxidase (LOX) is an extracellular matrix (ECM) enzyme that catalyzes the cross-linking of collagens or elastin in the extracellular compartment, thereby regulating the tensile strength of tissues. However, recent reports have demonstrated novel roles for LOX, including the ability to regulate gene transcription, motility/migration, and cell adhesion. These diverse functions have led researchers to hypothesize that LOX may have multiple roles affecting both extra- and intracellular cell function(s). Particularly noteworthy is aberrant LOX expression and activity that have been observed in various cancerous tissues and neoplastic cell lines. Both down and upregulation of LOX in tumor tissues and cancer cell lines have been described, suggesting a dual role for LOX as a tumor suppressor, as well as a metastasis promoter gene--creating a conundrum within the LOX research field. Here, we review the body of evidence on LOX gene expression, regulation, and function(s) in various cancer cell types and tissues, as well as stromal-tumor cell interactions. Lastly, we will examine putative mechanisms in which LOX facilitates breast cancer invasion and metastasis. Taken together, the literature demonstrates the increasingly important role(s) that LOX may play in regulating tumor progression and the necessity to elucidate its myriad mechanisms of action in order to identify potentially novel therapeutics.

A requirement for dimerization of HP1Hsalpha in suppression of breast cancer invasion.

The development and progression of cancer is controlled by gene expression, often regulated through chromatin packaging. Heterochromatin protein 1(Hsalpha) (HP1(Hsalpha)), one of three human HP1 family members, participates in heterochromatin formation and gene regulation. HP1(Hsalpha) possesses an amino-terminal chromodomain, which binds methylated lysine 9 of histone H3 (meK9 H3), and a carboxyl-terminal chromoshadow domain (CSD) that is required for dimerization and interaction with partner proteins. HP1(Hsalpha) is down-regulated in invasive metastatic breast cancer cells compared with poorly invasive nonmetastatic breast cancer cells. Expression of EGFP-HP1(Hsalpha) in highly invasive MDA-MB-231 cells causes a reduction in in vitro invasion, without affecting cell growth. Conversely, knock-down of HP1(Hsalpha) levels in the poorly invasive breast cancer cell line MCF-7 increased invasion, without affecting cell growth. To determine whether functions of the CSD were required for the regulation of invasion, mutant forms of HP1(Hsalpha) were expressed in MDA-MB-231 cells. A W174A mutation that disrupts interactions between HP1(Hsalpha) and PXVXL-containing partner proteins reduced invasion similar to that of the wild type protein. In contrast, an I165E mutation that disrupts dimerization of HP1(Hsalpha) did not decrease invasion. No gross changes in localization and abundance of HP1(Hsbeta), HP1(Hsgamma), and meK9 H3 were observed upon expression of wild type and mutant forms of HP1(Hsalpha) in MDA-MB-231 cells. Taken together, these data demonstrate that modulation of HP1(Hsalpha) alters the invasive potential of breast cancer cells through mechanisms requiring HP1 dimerization, but not interactions with PXVXL-containing proteins.