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Home , CDX1, Homeobox, HOXA13, HOXA3, HOXA4, HOXA7

ANTICANCER 27: 2141-2154 (2007)

Review Does -related "Positional" Genomic Information Contribute to Implantation of Metastatic Cells at Non-random Sites?

K.M. ANDERSON1, M. DARWEESH2, A. JAJAH2, P. TSUI1, P. GUINAN1 and M. RUBENSTEIN1

1Division of Cell Biology, Hektoen Medical Research Institute LLC, 627 S. Wood Street, Chicago; 2Division of Medical Oncology, Cook County Hospital, 1825 W. Harrison, Chicago, IL 60612, U.S.A.

Abstract. Reasons for the lodgment of metastases from Do HOX or Homeobox-related Contribute several types of solid cancer at apparently non-random sites Directly or Indirectly to Homing and Nidation of have not been established. Recently, a group of genes expressed Metastatic Cancer Cells? in human fibroblasts obtained from different anatomic locations was implicated in "positional" genomic information. Two basic models have been proposed to account for the Essentially, a Cartesian coordinate system identifying differential expression profile of fibroblasts located at fibroblasts originally resident at anterior or more posterior, different anatomical sites (1). In one model emphasizing the proximal or distal and dermal or non-dermal (heart, lung, etc.) persistence of embryonic "positioning", considerable innate locations was proposed. The determinants used for these in profiles of cognate cells located at comparable identifications included HOX genes, central to embryonic anatomical sites is predicted. In the second model, local segmental development, some of which are expressed in influences including epithelial-mesenchymal interactions are differentiated, post-embryonic cells. To the extent that HOX or considered to determine possible similarity. Of course, other homeobox genes are expressed in ectodermal, blending of these models might occur. mesodermal or endodermally-derived, malignantly transformed Rinn et al. (2) have provided further evidence for the former cells, they might contribute "positional" information to nidation hypothesis, in which the expression profile of fibroblasts at of specific malignant clones at non-random sites. As similar anatomical sites share features that identify the site understood in the past, interdiction of HOX or homeobox- from which they were harvested. Microarray related gene expression might reduce the probability of cancer profiles from 47 cultured human fibroblast samples, cell implantation or alter their destinations in complex ways. representing 43 different anatomical sites provided by 20 Ideally, by interfering with HOX or other homeobox gene- different donors were compared by a series of unsupervised related expression of antigenic determinants potentially and supervised hierarchical clustering studies. With successive contributing to their "homing" and nidation, reduced refinements, a group of 337 genes that included augmented implantation of circulating cancer cells could render them expression of functions associated with pattern and more susceptible to systemic or immunotherapy, intercellular matrix formation, cell to and that as demonstrated in mice. Furthermore, HOX or other included a number of HOX genes, was identified. With this homeobox genes or their products could provide novel intra- information, the original anatomical sites of the cultured or extracellular targets for therapy. fibroblasts could be distinguished as to anterior or posterior (caudal), proximal or distal and dermal or non-dermal (heart, lungs, prostate, intestines) location of the sampling. In view of their importance in embryonic , Correspondence to: K.M. Anderson, Division of Cell Biology, the HOX genes were further studied. Forty-two Hektoen Medical Research Institute LLC, 627 S. Wood Street, homeodomain genes that included 12 HOX genes reflected Chicago, IL 60612, U.S.A. Tel: +1 312 864 0564, Fax: +1 312 864 the identification of fibroblasts according to their original 9569, e-mail: [email protected] location, suggesting a HOX "code" for positional Key Words: Homeobox-related genes, metastasis, metastatic sites, identification. The genes expressed included HOXB2, B4, review. B5, B6, B7 and B9, limited to the trunk and non-dermal

0250-7005/2007 $2.00+.40 2141 ANTICANCER RESEARCH 27: 2141-2154 (2007) samples, while HOXD4 and D8 were expressed in trunk and malignantly transformed or not, are subject to "positional" proximal leg samples. HOXD4 was found by controls comparable to those of fibroblasts. There is immunoblotting in fetal lung fibroblasts but not in evidence that myogenic (9) and endothelial (10) cells exhibit fibroblasts from the foreskin. HOXA13 was expressed only some features of positioning, but additional evidence for in fibroblasts from distal sites, including the fingers, "instructional" contributions, especially during early foreskin, feet and prostate. Confirming studies were development between discrete epithelial-mesenchymal performed with RT-PCR of several HOX genes and interactions contributing to localization and induction of immunoblotting of HOXA13 from in vivo samples. Thus, specific cell types at different anatomic sites provides an fibroblasts from cognate sampling sites expressed greater alternate or additional explanation for these events (11). transcriptional similarity than fibroblasts from anatomically distant sites. These results were considered consistent with a Homeodomain and Homeobox Gene-related "positional" form of instruction of adult fibroblasts related Antigenic Determinants to embryonic expression and maintained by epigenetic mechanisms. Dorsal to ventral differences in In view of the many excellent reviews available (5, 12-21a,b), fibroblast gene expression present during embryonic we will not attempt to review the topic in detail, but a few development were not found, implying that epithelial general comments could be useful. In mice and men, the dictated dorsal to ventral fates in the limbs. HOX family includes 39 genes in 4 linkage groups (A Details of the anterio-posterior axis are complicated. through D) including some of the 13 homology (paralogue) Expression of the (en) gene in the posterior groups, from nine to eleven members in each linkage group compartment mandates (P) identity; its absence yields (A) located on 4 different , in order, A through D, identity in the anterior regions (3). The en gene can repress numbers 7, 17, 12 and 2. Human paralogue groups 1-8 are or activate target genes. P cells expressing the EN related to the (Antp) and groups 9-13 with are associated with hedghog (hh) activation. To achieve A-P abdominal-B (abd-B) of Home-C Drosophilia genes. A larger identities, en, a factor ci and hh are correctly expressed in group of non-HOX homeobox genes, of which HOX genes the required compartments and finally the Polycomb group are a subset, are more variable and located at diverse loci. (PcG) genes maintain en repression in all embryonic Class I HOX genes include 180 homeobox DNA segments, for example repressed hh in anterior segments. sequences, with a "signature" – ATTA – (and related -AT-) Left-right asymmetry across apparent boundaries has sequence(s), coding for 60 protein homeodomain been described for QL and QR neuronal cells in Chaenorh containing sequences complementary to appropriate abditis elegans (4). QL neuroblast lineages migrated promoters. HOX-related originate posteriorly, while QR neuroblast cells moved anteriorly, from the 39 HOX genes while the more numerous "class II" associated with HOX mab-5 gene expression in QL but not homeobox-containing non-HOX genes give rise to additional in QR cells, with an asymmetric response to a EGL-20/Wnt proteins, such as the TALE (three amino acid loop signal. A variety of mutants that disrupted these movements extension) superclass, including homeodomain-related were described. A basis for bilateral, right versus left proteins from genes denoted as PBC, MEIS, IRO, TGIF. symmetric positioning in chick depends upon sonic Class II homeobox genes give rise to additional hedgehog activating the bone morphogenic antagonist, homeodomain sequences and related proteins that have been Cerebrus protein, with expression of , activating the denoted as POU, LIM, PAX, NK-2, Hix, TCL, NEC, emsZF, homeobox gene, a member of the POU DNA-binding prd-like, prd, eve, lab, Dll, msh, NK-1, en, Abd-b Antp and domain family, and repression of the snail gene, cSnR (5). the TALE superclass (18, 19, 21, 22). Individual classes In QR cells, activin and its are expressed, Fgf8 is include families of related genes such as Antp containing activated, blocking expression of Cerebrus, allowing BMPO Dfd, Scr, Antp, Xiox and pd and TALE, mentioned above. to inhibit nodal, snail gene activity and repression of pitx2. This list is not in any sense meant to be complete and Similar events are considered to occur in human cells. additional divergent homeobox genes and regions of micro An additional concept is introduced in the HOX codes RNA have been identified. The approximately 200 identified believed to underlie and cell-specific development of homeobox-containing genes give rise to some 1000 related several organ systems, including the digestive tract (6), lung proteins identified in various species (13). Reference (13) (7) and hematopoietic system (8). Sequential, unimpeded includes the following sites for listings of HOX-like genes unfolding of HOX gene expression mandates the early (22a,b) and the National Human Research Institute normal growth and development of such complex organ homeodomain resource database (23). The HOX-Pro systems, while interference with these processes results in database (22a,b) available at their web site provides several various untoward disruptions of form and function. A very informative diagrams depicting reported interactions as further question is whether mammalian epithelial cells, genetic clusters, networks and maps between a number of

2142 Anderson et al: Homeobox-related “Positional” Genomic Information and Metastasis (Review)

Table I. Canonical sequence of early embryological events (4).

1. genes mRNAs specifying different regions of the fertilized egg with activation or inhibition of zygotic genes leading to 2. expression (their lack = a gap in segmentation) followed by 3. Regulation of pair-rule genes, mandate periaxial segmentation, activating 4. Segment polarity genes (e.g., hedgehog) leading to…. 5. expression…..and 6. Realisator gene and regulatory molecule-expression.

Events 1 through 3 occur in a syncytium; homeobox and HOX gene expression and cell fates are established during stages 4 to 6 and following. Continued expression of some homeobox genes are evidently necessary for maintenance of post-embryonic cellular growth and differentiation.

HOX genes as these are affected by bone morphogenic post-embryonic expression of HOX genes is much less proteins 2 and 4, Krox-20, Pbx-1, AP-2, Kraisler, retinoic acid organized, more "individualistic". In mammalian , via RAR and RXR and (probably) B-FGF. HOX genes can be expressed or inhibited, compared to Early expression of HOX and other homeobox genes are control cells; a few examples are presented in Table II. Five major determinants of embryonic segmental and antero- out of 39 HOX genes were expressed in normal , posterior organization of the basic body structure, preceded and 3 (HOXA7, A9 and C6) at significantly higher during embryogenesis by a sequence of events defined as concentrations in squamous cell carcinomas (24). Eight other maternal gene effects, expression of GAP genes and HOX genes (HOXA10, 13 B7, C4, C8, D9, D10, D13) were regulating pair-rule genes which divide the into peri- only expressed in the malignant cells. HOX C4, 5, 6 and 8 axial segments, followed by segmental polarity expression in were overexpressed in malignant human prostate cell lines, an antero-posterior axis within the segments which establish primary tumor cells and lymph node metastases (25). cell fates and followed by expression of homeotic "selector" Reduced expression (except for HOXC11) of numerous HOX and the downstream "realisator" gene expression (4 and genes was found in human invasive breast cancer (26). Table I). The first three events are involved in activation of Overexpression of HOXA1 in mammary carcinomas HOX and other homeotic genes leading to multiple increased cell anchorage-independent cell division, Bcl-2 functionalities mentioned above. "Selector" genes include expression, resistance to daunorubicin and caused neoplastic HOX genes and "realisator" components populating the transformation (27). pathways and implementing pathways. HOXA5 expression induced in MCF-7 cells Homeobox transcription factors are implicated in cell that involved 2 and 8 (28). Down-regulation of division, adhesion, migration, forward and retrograde HOX, MEIS1 and MLL (mixed lineage leukemia) during differentiation and , leading to normal hematopoiesis was absent in acute myelogenous component molecules contributing to these states. leukemia cells and expression of HOXA9 and 10 and B3 These abbreviated comments, based on our understanding and 4 was greatly reduced in cDNA from CD34– compared of the literature we have read, are intended only to suggest with CD34+CD38– normal cells (29). Chromosomal re- the evident complexity of a few of the HOX and homeobox arrangements of HOXA9 or HOXD13 fused with NUP98, interactions identified by the experts who actually study them, an activator of HOX gene expression, was present in some to suggest their potential contribution to "positional", possibly acute myeloid leukemia cells (30, 31) and gene expression non-random genomic information or as subsequently profiling of myeloid leukemias identified 6 prognostic transpired, even more generally to additional features of the groups, one of which, cluster D, underexpressed HOXA9 "metastatic cascade" that might be co-opted during malignant and A10 (32). HOX gene expression in primary and transformation or subsequent metastasis. metastatic cancers, cited below, can be similar or differ. Many unresolved questions of interest include: What are Primary and Metastatic Cancer Cells Modulate the the functions of HOX (and homeobox) genes that continue Expression of Various HOX Genes to be modulated in post-embryonic cells? How do these functions differ from their temporally and spatially A number of homeobox genes are expressed in differentiated integrated expression during embryological development? normal (5-7, 9, 10) and in malignantly transformed cells (15- Is the concept of – different functions of a gene 18, 21a,b; Table II). During embryological development, HOX in different contexts – germane to understanding the gene expression occurs along an anterior to posterior, 3’ to 5’, consequences of post-natal expression and are any of these spatial and temporal direction; this is not the case of functions co-opted during malignant transformation, as expression between paralogue members (13). Presumably, seems to occur during leukemogenesis (30-32)?

2143 ANTICANCER RESEARCH 27: 2141-2154 (2007)

Table II. Expression of HOX genes in some representative human cancers and derived cell lines compared to normal fibroblasts.

Leukemias: HOXA9 or HOXD13 translocations with NUP98 (AML); MLL and HOX expression (ALL, AML); HOXA4 to HOXA11, down-regulation of HOXA, B, C with lineage differentiation: +HOXA7 (AML), +HOXA9,10, B3, B4, MEIS1, MML (AML), +HOXC4, 5, (6, non Hodgkin lymphomas) Breast cancer: +HOXA1, –HOXA10, +HOXB7, –HOXA5; – (HOXA1-3, A5, 9, C11, D3, 4, 8, 9,10, 13), +HOXC11; –HOXC6: HOXD10, D13 Lung cancer: +Wnt7A and HOX; HOXD3; +HOXA1, A5, A10, C6, +D9, 10, 11 (squamous); +HOXA5, A10 (adenoca); +HOXD3, SCLC, –(HOXB and C loci, lower in metastatic ca); SCLC, +HOXA7, A9 C 4, 5, 6, –HOXC5, D13; NSCLC, +HOXA7, C5, D13; – HOXC4, C6 : +HOXA10, 13, B7, C4, C8, D9, D10, D13 Colon cancer: HOXB6, B8, HOXC9, various Prostate: +HOX C4, 5, 6 and 8; –HOX B13; –HOXD10 Renal cancer: HOXA9; rarely +HOXD10, HOXC9; –HOXA2, E2, +HOXH3; group 10 (HOX1D, 2F, 3E, 4B differ from control); +HOXC11; –HOXA2, E2, +HOXH3 Bladder cancer: +HOXC4, 5, 6, C11 Cervical cancer: +(HOXA1, B2, B4, C5, C8, C10, D13 in 7 to 11 lines, absent in normal cells) Ovarian cancer: +HOXA9, 10, 11; +HOXB7; HOXA7 Endometrial cancer: –HOXA10 Melanomas: HOXA1, 2, C4, B13 (metastases); HOXA11,13, B9, D12,13 > naevus; HOXB7; –HOXD3; HOXC10,11,13 alter metastatic potential. Thyroid: HOXD9, not in ca Astrocytes: HOXA13, B13, D4, D9, D10, D13, differential expression, cell lines and cancers

"Control" expression of HOX genes in cultured fibroblasts (1, 2) related to position. HOXA10, 11, 13 proximal/distal upper forelimb, HOXA10 and 13, distal toe and foreskin; HOXB2, 4, 5, 6, 7, 9 (trunk and non-dermal); HOXC5, upper limb; HOXD4 and D8, trunk and proximal leg segments. Increased unless denoted as - or decreased expression. These representative examples are in no way exhaustive and most are cited in the references provided. Expression of HOXA10, 11, B2, 4, 6, 7, HOXC5, D4 and D8 is altered in representative cells from one or more types of cancer. Although members of 3 different HOX classes are expressed in normal fibroblasts from several locations and in various human cancer cells, generally from primary sites, no compelling paralogous ( clustered 1-13) HOX gene expression is revealed from this very limited data-set based on the references cited. Data from class 2 homeotic genes seems to be even more limited but could possibly prove relevant to "positioning". Increase or reduction in paralogous HOX gene expression and characteristic metastatic behavior is not available. We did not attempt to make an extensive survey of all available HOX and non-HOX homeobox gene expression in cancers.

The Epithelial-ªesenchymal Transition and the the epithelial to mesenchymal transition of human breast (36) Argument Restated and uterine cancer cells (37), respectively. In the former, HOXB7 overexpression in primary (x3) and metastatic (x18) The epithelial-mesenchymal transition (EMT) is considered to cancer cells was examined in transfected culture lines, be fundamental to embryogenesis and some of its properties identifying increased bFGF synthesis, greater Ras and RhoA are exhibited in the behavior of metastatic cancer cells. These protein activity and phosphorylation of MAPK extracellular include changes in cell morphology, increased resistance to kinases, reversible by inhibitors, and overall implicating a programmed cell death as exemplified by apoptosis, reduced HOXB7-induced bFGF – Ras / Rho pathway in EMT. Down- cell to cell adhesion with an ability to migrate, functional regulation of HOXA10 in endometrial carcinoma correlated capabilities associated with homeobox, including HOX gene with less differentiation and increased methylation of its expression, which conceivably could be co-opted in some promotor. Increased expression of HOXA10 in cultured cells manner and contribute to the "metastatic cascade", including reduced their invasive properties; this was related to lesser elements responsible for cancer cell "homing", among other expression of Snail, allowing increased expression of E-cadherin properties (5, 11, 33, 34). In a number of experimental systems, with increased cell adhesion (37). these characteristics have been associated with increased In organisms capable of , such as salamanders, if expression of N rather than E-cadherin, of vimentin, ECM an amputated limb is rotated 180 degrees and re-implanted, proteins, focal contact proteins, different , nuclear supernumerary digits in mirror-image symmetry are induced; localization of B-catenin and increased synthesis of local cells in both developing and regenerating limb buds retain transcription factors including Snail 1 and 2, Trist, E 47 among similar instructions (5). A recent comparison of gene expression others that inhibit formation of E-cadherin. Malignant prostate in mammalian wound healing and renal cell carcinoma found cancers undergo a transition from an E-cadherin to the 77 percent of genes examined were concordantly expressed and mesenchymal M form, exhibited a more aggressive clinical 23 percent discordantly expressed, the latter defined as altered behavior (35). HOXB7 and HOXA10 have been implicated in in opposite directions (38). Proliferation and immune responses

2144 Anderson et al: Homeobox-related “Positional” Genomic Information and Metastasis (Review) characterized the former group and altered , or other mechanism of osteocalcin activation contributed to glycolysis, including hypoxia-inducible factor and insulin-like by activation of specific homeodomain genes active in -I were present in the latter. The extent of metastatic cancer cells could in principle, contribute to overlapping functional categories (39) between the epithelial- induction of osteoblastic lesions. HOXA9 represses TGHF-‚- mesenchymal transition (5, 11, 36, 37, 40), the wound healing- induced osteopontin gene transcription via smads; 4 was response (38) and established cancers, reflected in their a common signaling component for both BMP and the TGF-‚ respective gene profiles (e.g., 41) did not include prominent pathways (48), activation of homeobox genes at the HOXC expression of HOX genes. In DNA microarray studies, by induced interactions with growth factors, epithelial cancer progression, employing mouse mammary inflammatory and adhesion molecules has been EpH4 cells and several Ha-Ras-transformed variants, HOX8.1 reported (49), while bone extracellular matrix was reported to (Msx2) and several agents able to affecting HOX gene activate HOX genes, identified by multiple ATTA motifs in expression during embryogenesis (BMP-4, KROX20 and un-translated 3’ regions, in human LNcap prostate cancer cells 24 (EGR-2 and -1)), Kruppel-like factor (LKLF) and RAR-a) cultured in the absence of androgens (50). LNcap cells studied were represented in the epithelial to mesenchymal transition in nude mice as LNcap/fetal fibroblast chimeric tumors that (40). Identified genes were grouped into 10 up-regulated and 9 were androgen-independent all expressed HOX genes. down-regulated categories while some 15% of all regulated There is some disagreement concerning the importance genes were found to be controlled at the translational level, as of this transition in clinical cancer, based upon factors such determined with polysome-bound mRNA expression profiling. as a lack of morphological differences between primary and Signal transduction and implementing pathways detected in secondary deposits, the presence in some metastases of these and other studies of EMT, including wnts, Mets, src, E- rather than M-cadherin, and the lack of EMT in , sox, PI3K/AKT, kinases, RAS, EGF, tumors (51). The presence of changes consistent with EMT HGF and TGF-‚ (42) might have been activated, at least in at the invading front of metastases, numerous biochemical part by prior homeobox, including HOX gene transcription. In events associated with the EMT and the ability of metastatic a study of CD34+ human cord blood cells transduced with cells to undergo a reverse differentiation from M- to HOXA9 or HOXA10, genes in the Wnt pathway including E-like morphology and , exhibiting reduced Wnt10B, Wnt receptors 1 and 5,v-ets-related (ERG), invasiveness seem to counter the objections (33). Iroquois 3 (IRX3), aldehyde dehydrogenase-1 and a long chain acyl-CoA synthetase were up-regulated while HOXA10 Factors Affecting Dissemination and Nidation of repressed several and globin-related genes associated Cancer Cells with differentiation; otherwise the effects on transcription of the two genes largely overlapped (43). Downstream To metastasize, malignantly transformed cells must successfully modulation of genes related to energy , negotiate a metastatic cascade, encompassing local invasion growth, division and differentiation by HOX genes situated from the primary, escape into the circulation, transport and them at points of fundamental cellular control. The concept of arrest at distant sites, extravasation with local invasion and pleiotropy in which a gene may play a different role in different growth, including induction of (34). To abstract cells or in the same cell or cell lineage at different times may and grossly oversimplify, the majority of the relatively few apply to both embryogenesis and to carcinogenesis and migratory cancer cells that successfully implant initially require subsequent release, homing or nidation of transformed cells. available routes for hematogenous or lymphatic spread (34, 52- Metastases from several major epithelial cancers, including 54). During embryogenesis, the EMT transition is considered prostate or breast cancers to bone and instances of ipsilateral contributing to the migratory and invasive properties cancer metastases occur more often at characteristic, non- characteristic of mesenchymal cells (5) and of metastatic random sites than would be predicted by chance (34, 44-46). cancer cells. There is evidence that the ability of some For prostate cancers metastasizing along the axial , malignant cells to colonize a distant site can depend upon the para-vertebral venous plexus has been considered unique properties of the migrating cells themselves, and less contributory; alternately a clone of prostate cancer cells on the properties of the site. Human colon cancers exhibiting exhibiting expression of a co-opted or otherwise dysfunctional metastatic (M) or non-metastatic (N) properties in a host were HOX (or homeobox)-related gene contributing during transplanted to the colon or liver of nude mice (55). Both M embryological development to anterior-posterior or dextral- and N cells invaded the colon; in contact with mouse liver, only sinistral or even superficial versus deep "positioning" could be M cells were invasive. This was considered dependent upon suggested as contributory. For example, osteocalcin, whose properties inherent in and unique to M cancer cells. expression is restricted to osteoblasts, which it activates, is Unidentified exchanges of metabolic or immunologic signaling regulated by homeodomain factors, AP-1 related proteins and between the M cell seed and mouse liver soil might have other bone-restricted transcription factors (47). A paracrine existed. Possibly N cells were unable to induce some hepatic

2145 ANTICANCER RESEARCH 27: 2141-2154 (2007) event necessary for their nidation or the liver lacked a HOX-related Responses that Could Contribute component required for a potential N-cell invasion that was Directly or Indirectly to Proximal or Distal irrelevant to or provided for invasion by M cells. Features of Cancer Cell Homing and Nidation Two major ideas underlying metastatic specificity entail either a homing explanation dependent upon the migratory Several descriptions of gene profiles associated with cancer cell, the primacy of the soil relative to the cancer cell metastases from non-hematological cancers do not seem to seed, or a blending of the two (56). In either case, cancer include primary modulation of HOX genes (67a,b, except c to cells are thought to be engaged via a complementary 70, to cite several, but see ref. 43; HOXA9 and A10 in normal interaction between adhesion receptors associated cord blood cells; 31, 32 and leukemia and 67c, increased with/without secretion of chemotactic factors (57). An HOXA5 in post-chemotherapy versus primary chemosensitive example of a second means of interaction is provided by the ovarian cancers). The molecular signature of breast cancer role of the stromal cell-derived CXCR4 pathway in prostate metastases to bone marrow involved repression of genes cancer metastatic to bone (58). In breast cancer metastases, associated with extracellular remodeling, adhesion, receptors, such as CXCR4, CCR7 and their cytoskeletal changes and signal transduction, emphasizing on breast cancer cells, a parathyroid-related RAS and Hif-1a, when compared with lymphatic metastases (PTHrP) found in osteolytic lesions or -1 and (67a). Comparison of normal and malignant colon resulted in platelet derived growth factor in osteoblastic lesions can be identifying 584 genes whose expression between them differed contributory (59). As reviewed in (60), HOX2A inhibits (68). Major differences in genes concerned with proliferation, cartilage condensation and bone formation by inhibiting apoptosis and the immune response were emphasized, e.g. Sox9 expression and down-regulating Cdfa1; HOXD11 and Pi3K, ELF4eL3, CASP3, HLA-E and B2-m, in addition to D13 reduce cartilage formation and length of the tibia and numerous others. L’Esperance et al. found that 121 genes fibula; HOXA13 inhibits cartilage proliferation; HOXD and were often up-regulated and 54 genes down-regulated in post- BMP-2 are concerned with anterior to posterior limb chemotherapy-treated ovarian cancer samples, compared with positioning and in the hindgut BMP-4 and Abd-B-like the primary tumors (69). Up-regulated genes included a (paralog 9-13) HOX proteins function as downstream number concerned with chemoresistance, those down- modulators of sonic hedgehog signaling. Bone morphogenic regulated with chemosensitivity, proliferation, cell cycle proteins can regulate HOX gene expression and the latter control, tumor suppression and apoptosis. Genes associated can regulate expression of the former (60). For example, with diverse metastatic human adenocarcinomas include Smad 1, responding to BMP, displaces HOXC8 from the metastasis suppressor genes Nm23, KiSS1, KA11, CAD 1, and activates osteopontin and osteoprotegerin BRMS1 and MKK4 (70). Comparison of gene expression gene transcription. Smad 6 serves as a co- of patterns from a variety of cancers identified 128 genes that HOXC8. Additional details are provided in (60). could be refined to include 17, comprising 8 up-regulated and The developing concept of a pre-metastatic niche 9 reduced that distinguished primary and metastatic including locally increased , increased bone adenocarcinomas. An analysis of 13,000 genes in 11 breast and marrow-derived cells expressing vascular endothelial growth 11 colorectal cancers identified a number of cancer-related factor receptor 1 and metalloproteinase activity at a site genes separated into nine categories such as cell adhesion and prior to the appearance of migratory cancer cells, in motility, signal transduction, transcriptional regulation (71). response to growth factors elaborated by the primary cancer HOXA3, categorized as a regulator of transcription, was the (61, 62). Treatment of tumors with agents that only HOX gene included in this classification. A number of disrupt the vascular system resulted in mobilization of bone other cancer-related genes were denoted; conceivably some marrow-derived circulating endothelial cells able to colonize non-HOX homeobox genes or associated pathways or the cancer, which anti-angiogenic could at least components related to them are represented. partially inhibit (62). It does appear that so far the most direct association Genes that inhibit cancer metastases without necessarily between HOX gene expression and related properties is altering malignant transformation represent another presented by normal hematopoietic development and acute potential signal mechanism that might be influenced by leukemia. Lacking much information about the HOX or other homeobox gene modulation (63-65). For consequences of modulating (often inhibiting) HOX gene example, inhibition of Twist, an important regulator of expression, with their largely unknown (in much detail) morphogenesis and inducer of the EMT, with siRNA, consequences of functioning as transcription factors, let reduced the number of tumor cells in the circulation of mice alone possible effects of non-HOX homeobox genes, it is not and subsequent metastatic lung cancers (66). High surprising that identifying precise contributions to malignant concentrations of twist in human lobular breast carcinomas cellular behavior in solid cancers, especially any with correlated with invasiveness. therapeutic applications, has been difficult. It may be that

2146 Anderson et al: Homeobox-related “Positional” Genomic Information and Metastasis (Review) the question is not whether HOX genes are demonstrably prevented metastases and reduced the size of the up-regulated, but rather that during post-embryonic transplanted primaries (78). Sonic hedgehog increases malignant transformation, specific HOX genes are turned on ectopic expression of BMP-4 and specific HOXD gene but do not achieve the value selected to define significantly expression, albeit in the chick (79), and has been implicated increased expression in microarray studies, turned off or in early and later events underlying the development of even regulated post-transcriptionally, having initiated events pancreatic cancer (80). In the latter paper, increased that cannot be easily shown to have required HOX gene expression of the denoted as "glil" expression. Simple feedback or even reverberating circuitry caused a reduction in E-cadherin and an augmented EMT. is not difficult to model (15). While examples of a precise assignment of positional If the question is broadened to include the expression of specificity in cancer cells due to HOX or homeobox-related non-HOX homeobox genes (e.g., 72, 73a,b), apparently not transcription factors seem lacking, given the ancient and all of which function as transcription factors, a more robust central role for many of these genes in organizing the argument might be developed. Genes activated by the mammalian , and the possibility of their co-option homeodomain BARX2 protein studied in the MCF-7 breast during malignant transformation, considerable scope for cancer cell line by immuno-precipitation and other oncological mischief seems implied. A few additional techniques identified 60 potential candidates that included examples follow, the universality and implications of which transcription, receptor, , cytoskeletal, CAM, signaling are often not clear. and activities, and others outside these categories (72). RNAi partially inhibited a number of genes related to Earlier (proximal) events. During HL-60 monocyte growth and invasiveness of MCF-7 cells, and indicated differentiation, down-regulation of HOXA7 was necessary for coordination of several differentiation pathways. Some of the cell adhesion and migration on fibronectin. HOXA7 is often hundreds of genes identified (references 67-71, as up-regulated in acute myeloid leukemia, and its over-expression representative examples) may have included components of in HL-60 cells interfered with these interactions (81). Up- pathways related to antecedent HOX (or homeobox) gene regulation of HOXA5 induced apoptosis in breast cancer cells expression. An extensive survey of homeobox expression in that was mediated by caspases 2 and 8 (28); up-regulation of cancer cells is complicated by variations in nomenclature, e.g. the HOXA1 homeobox gene induced increased proliferation, PDX-1 or pancreas-duodenum homeobox-1 has also been resistance to doxorubicin and malignantly transformed denoted insulin promotor factor-1, islet/duodenum immortalized human mammary cells. Enforced overexpression homeobox-1, somatostatin transactivating factor-1, insulin of HOXA1 promoted invasion in in vitro and in vivo studies in upstream factor-1 and glucose sensitive factor (74a,b). nude mice. Over-expressed HOXB7 promoted the epithelial to Factors affecting normal lympho-hematopoietic cell mesenchymal transition of human breast cancer cells (36). adhesion and homing, including participation of integrins, Transcriptional repression of a limited area of the HOXA gene immunoglobulins, lectins, sialomucins, hyaladhedrin and cluster in human breast cancer was due to aberrant DNA CD 44, 38, 144 and a number of including methylation (82) and associated with reduced expression of the CXCR1-5, CCR1-10, among others, have been identified HOXA without evidence of consistent changes in (75). Inappropriate HOX and homeobox gene expression in paralogous HOXB,C and D clusters (26). cancer cells might contribute to particular mechanisms for HOB7 is underexpressed in some breast cancer metastases, metastatic homing; e.g. neural cell adhesion molecule while reduced HOXA5 expression in some breast cancers is promotor activity was enhanced by co-transfection with associated with limited expression (83) and down- HOX –2.4 and 2.5 (76). Early contributions of HOX gene regulation due to methylation of HOXA10 in endometrial expression to the metastatic cascade may be necessary but cancers was associated with increased tumor grade (37). themselves insufficient to mandate nidation. In primary and metastatic melanomas and 25 melanoma Despite earlier interest in these matters, in general, cell lines, HOXB7 constitutively activated fibroblast growth studies of homeobox and HOX gene expression do not factor (83). Others noted expression of HOXA1, A2, C4 and seem to have led to many potentially exploitable insights, B13 in distant melanoma metastases, without a relationship even with the demonstration of differences in HOX gene to their sites (84). HOXB7 was overexpressed in human expression between the primary and metastatic cells (77). ovarian carcinoma cells, and increased the intracellular However, more recent evidence of their participation in the accumulation of FGF, a ligand with angiogenic and development of pancreatic cancer implies a paradigm for mitogenic activities (86). The antigenic product of HOXB7 contributions to malignant transformation in other cancers served as a growth factor for ovarian carcinoma cells, (78). Blocking sonic hedgehog with cyclopamine reduced increasing the intracellular concentration of FGF. Down- the invasiveness of human pancreatic cancer cells regulation due to methylation of HOXA10 in endometrial implanted in nude mice, and combined with gemcitabine cancers was associated with increased tumor grade (37).

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In a number of experimental systems, HOX genes can induced SMAD phosphorylation of pre-osteoblast MC3T3 cells promote or retard angiogenesis, depending upon the (105). Prostate-conditioned medium induced mineralization of circumstances (87). In addition to a number of homeobox- these cells. Anti-BMP-6 medium reduced LuCap 23.1-induced related genes, HOXD3 likely promotes invasive/migratory osteoblastic activity of human fetal bone implanted in nude behavior and HOXB3 capillary morphogenesis of endothelial mice but left osteolytic activity unaffected. Intraosseous but not precursors as a response to angiogenic signals (88), HOXD10 subcutaneous tumor sizes diminished. BMP-2, -4, -6 and -7 had suppressed angiogenesis of human microvascular cells (89), no direct effect on prostate cell growth but BMP-2 and -6 HOXA5 promoted while HOXD5 and D10 down-regulated increased their invasive activity; serum endothelin-1 levels did angiogenesis, the former by suppressing pro-angiogenic genes not differ among the groups studied. including VEGFR2, A1, Hif1a and up-regulating thrombospondin-2 (90). Overexpressed HOXD3 induced a Several more specific biochemical mechanisms implicated in HOX coordinate expression of metastasis-related genes with altered gene expression. A number of HOXC8 candidate target genes adhesion molecule and metalloproteinase activity, increased have been identified in mice overexpressing the gene (106). motility and invasiveness in human A549 lung cancer cells (91) Based on oligonucleotide microarray data from C57BL/6J that implicated TGF-‚ dependent and independent pathways mouse embryo fibroblasts, 34 genes were altered at least 2-fold, (92), while transduction of antisense into human melanoma 16 up- and 18 down-regulated. Expression of osteopontine was cells reduced these properties; control cells did not express reduced and frizzled homolog 2 up-regulated, both about 4-fold HOXD3 (93). Expression of E-cadherin and plakoglobulin and an interaction between the OPN promoter and HOXC8 declined, alpha3 and beta3, matrix metalloproteinase- demonstrated by chromatin immuno-precipitation. Overall, 2 and urokinase-plasminogen increased, and integrin and N- genes involved in apoptosis and in cell motility and proliferation cadherin were expressed. Blocking integrins with anti-integrins were down-regulated, adhesion molecules mixed, increased yielded increased haptotaxis to fibronectin. Homeobox D10 expression of frizzled, a for the WNT-B- can phenotypically revert breast tumor cells (94). catenin-TCF-signaling pathway important in several cancers, Overexpression of HOXC8 in prostate cancer was correlated the potential utility of osteopontine as a marker for a number of with loss of differentiation (95). HOXB13-derived protein cancers supported and findings generally consistent with an inhibits androgen-receptor-positive prostate cancer cells by important role for HOXC8 expression in neoplastic interaction with the receptor itself (96). An extensive list of development and further were presented. including , , , Targets of HOX genes in developing murine hindbrain and vitamin D and especially retinoic acid via RAR and RXR spinal cord, reviewed in (13), include discussions of cell receptors modulate HOX gene expression (97). adhesion molecules (N-CAM, L-CAM, cadherin 6, a-2-integrin, E-cadherin, aIIb33, CD44, implicating HOXA1, HOXC8, Distal (nidation) events. Examples in which HOX genes HOXA9, the latter two inhibiting osteopontine transcription promote the EMT (36, 37) and modulate expression of specific and themselves inhibited by Smads and their signaling in the integrins, cadherins, caveolins and other adhesion factors (98- TGF-‚ and bone morphogenic pathway); the potential tumor 100) provide the most direct mechanisms for potential homing suppressor mgl-1, a target of HOXC8; ephrin receptors, an to distant sites. Inhibition of caveolin in human breast cancers IgCAM subfamily of receptor tyrosine kinases (RTK) expressed exhibiting suppressed HOXA expression (82) is a recent in rhombomeric patterns coordinately expressed with HOXA1, example. Caveolin, a protein component of caveolae, the non- HOXB1 and HOXA2 with another ephrin, HOXA5 and clathrin coated invaginations of plasma membranes, inhibits HOXB7 and the G-protein pathway via Purkinje cell-specific anchorage-dependent growth and the invasiveness of human pcp-2 inhibited by engrailed-2 (EN-2) but activated by the breast cancer cells (101) Evidence that some primary cancers HOX genes post-natally, HOXB4 and the GTPase Ras can themselves evoke complementary responses at remote sites superfamily member, Rap1, antagonizing Ras signaling of the contributing to nidation further complicates this issue (61, 62). ERK/MAPK pathway; protease inhibitors including serpins and Homeobox A9 regulates transcription of the EphB4 receptor HOXB5; transcription factors involved in HOX transactivations which affects endothelial cell movement and vessel formation such as Otx homeobox genes and HOXB1, B2, B3, the Iroquois (102), while the element in EphA2 is activated by gene encoding TALE homeodomain proteins, downstream HOXA1 and B1 proteins (103). HOX13 regulates the from HOXB4. The extent to which these associations are expression of bone morphogenetic proteins 2 and 7 by unique to the or have some applicability to association with their enhancer regions, indicated by immuno- other organ systems has probably not been established. precipitation studies and partial correction of HOXA13 mutant As cited in (43), in human cord blood stem cells, HOX after addition of BMP-2 or -7 (104). Bone A9 and A10 regulate genes involved with cell proliferation, morphogenetic protein-6, commonly present in prostate including ALDH1, Iroquois 3 (IRX3), ets-related gene cancers, promoted osteoblastic metastases associated with (ERG), very long chain acyl CoA synthetase with influence

2148 Anderson et al: Homeobox-related “Positional” Genomic Information and Metastasis (Review) on lipid, energy and ion metabolism, dehydrogenase effect and whether HOX or homeobox genes might contribute and retinal dehydrogenase and the wnts pathway with simply has not been determined. A sufficiently extensive effects on B-catenin-TCF, c-jun-N-terminal kinase (JNK) analysis of even the available data from multiple sources might and the Ca2+ and cGMP pathways. Lastly, HOX 11 suggest linkages between homeobox gene expression and interacts with protein phosphatases PP2A and PP1, metastatic sites by particular cancer cell clones. interrupting a G2/M checkpoint (107). Until the functions of several hundred homeobox genes, some 1,000 homeodomain proteins in various species and their Conclusion interactions with the rest of their respective genomes are more fully understood, conclusions regarding putative effects of HOX Initially we wondered if HOX or homeobox-related gene and homeobox genes on the metastatic cascade, based on the expression influenced homing and nidation of non- available information and related inferences optomistically hematopoietic, solid malignancies to non-random sites. As might support the Scottish legal verdict of "not proven". information accumulated, additional influences on the events It seems surprising that the impact on cancer biology of ascribed to the metastatic cascade had to be included. homeotic genes, exerting such central effects on cell biology, Provisionally, there appears to be no common pattern to has not been more profound. Considering the cyclic activated or inhibited HOX gene expression, no simple involvement of multiple signaling genes and their functional identity with those reported present in fibroblasts (1, 2) or any expression including FGF, notch, wnt and others mentioned proven relation with non-random metastases; at least we did above, during for example, mouse segmentation (109), the not encounter such reports from our albeit limited search biochemical substrata for central effects on mammalian cell (Table II). To expect to identify such a simple correlation behavior, especially were such genes inappropriately activated would probably have been surprising and actually or active ones inhibited in some disorganized manner, these unreasonable, given the limited information regarding and others should provide many potential sites for oncological downstream modulation of pathways by HOX or other mischief. Particular components of these networks and homeobox gene-related transcription or the control of these pathways – their "kernels", "plug-ins" and subroutines (15) – genes and any directly or indirectly related proteins. Some might be susceptible to co-option during carcinogenesis, and cancers share modulated expression with normal fibroblasts by their modulation confer properties on transforming cells of HOXA10, A11, HOXB2, 4, 6, 7, HOXC5 and HOXD4 and associated with clinical malignancy, as has been understood in 8, activities which are increased or decreased compared with general terms for some time. Attempts directly to inhibit an controls. In view of their potential functions, co-expression of overexpressed HOX gene might be partially thwarted by their several classes of HOX genes in normal fibroblasts and in functional redundancy, expression in normal cell growth and examples of human cancers is of interest, but lacking sufficient development or in initiating self-sustaining regulatory circuits knowledge of the consequences from modulated HOX and of no longer requiring their active participation. homeobox gene expression, support for causal relationships To the extent that modulation of specific cancer cell-related between such comparisons and any consequences for cellular HOX and homeobox-related gene expression altered or even "positioning" remains suggestive but elusive. prevented homing and nidation of circulating cancer cells at From their embryological antecedents, one might have random or non-random sites, a prolonged exposure to anticipated metastases influenced by some HOX genes systemic immuno- or chemotherapy should be more lethal. exhibiting "positional" information distributed in segmental The combination of gemcitabine and blockade of sonic patterns. Ipsilateral metastases in the absence of other hedgehog with cyclopamine in pancreatic cancer implanted in affected sites is consistent with "positional" instructions male athymic mice represents a new and innovative approach related to anterior / posterior and dextral / sistral location, to treatment of cancer directed against a class of control while dorsal / ventral positioning of cells occurs during the molecules not frequently examined as potential therapeutic expression of various HOX codes (5-8). HOX genes at the targets that can influence other regulatory factors such as 3’ end can influence those in the preceding 5’ paralogue, but bone morphogenetic proteins and HOX genes (76, 78-80, 93, generally posterior genes are said to be expressed to a 94). Increasing melanoma cell susceptibility to CTL-mediated greater extent than more anterior ones. lysis by increasing ICAM-1 expression (110), increasing The more numerous non-HOX homeobox genes do not -dependent cellular killing with anti- E-cadherin seem to have been systematically surveyed for "positional" or antibody (111), or disrupting cell-cell interactions by inhibiting other comparisons between fibroblasts and cancers. For av integrins thus increasing antitumor and anti-angiogenesis example, overexpression non-HOX homeotic genes Cdx1 and activity in vivo have been reported (112). More recently, Cdx2 promoted the malignancy of colon cancer cells, expressing blockade of VEGF combined with GM-CSF tumor cell a tumor-suppressor effect in the adult colon but a homeotic role immunity prolonged survival of B16 melanoma and CT26 during development (108). The mechanism of the suppressive colon carcinoma cells in tumor-bearing mice (113).

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A related benefit of interfering with homing/nidation 9 Donoghue MJ, Morris-Valero R, Johnson YR et al: concerns the cancer stem cells that presumably would not be Mammalian muscle cells bear a cell-autonomous, heritable able to implant. Interest is being focused on the role of memory of their rostrocaudal position. Cell 69: 67-77, 1992. 10 Chi JT, Chang HY, Haraldsen G et al: Endothelial cell cancer stem cells as the correct target for therapy of primary diversity revealed by global expression profiling. Proc Natl or metastatic cancer (114) and the development of cancer Acad Sci USA 100: 10623-10628, 2003. stem cells in hypoxic environments as major sources of 11 Shook D and Keller R: Mechanisms, mechanics and function therapeutic resistance (115). For almost all solid cancers, of epithelial-mesenchymal transitions in early development. cancer stem cells in locations shielded from therapeutic Mech Devel 120: 1351-1383, 2003. agents, especially in regions of hypoxia, are thought to 12 Pearson JC, Lemons D and McGinnis W: Modulating Hox circumvent systemic therapies. Inhibiting nidation should gene functions during animal body pattering. Nat Rev Genetics reduce the number of stem cells in hypoxic or other protected 6: 893-904, 2005. 13 Akin ZN and Nazarali AJ: HOX genes and their candidate regions of a metastasis. Release of non-replicative, more downstream targets in the developing central nervous system. differentiated cells from a primary cancer that successfully Cell Mol Neurobiol 25: 697-741, 2005. lodge at some site and dedifferentiate to gain the ability to 14 Lemons D and McGinnis W: Genomic evolution of Hox gene replicate seems less likely. clusters. Science 313: 1918-1922, 2006. Accumulation of a more complete repertoire of HOX and 15 Davidson EH: The Regulatory Genome. Elsevier, Amsterdam, homeobox transcription factor responses and the pathways AP, pp. 223-240, 2006. they modulate will likely provide important insights into the 16 Lappin TRJ, Grier DG, Thompson A and Halliday HL: Hox interpenetration of developmental processes as some of genes: Seductive science, mysterious mechanisms. Ulster Med J 75: 23-31, 2006. them may reappear in the biology of cancer cells. 17 Cillo C, Cantile M, Faiella A and Boncinelli E: Homeobox genes in normal and malignant cells. J Cell Physiol 188: 161- Acknowledgements 169, 2001. 18 Maroulakou IG and Spyropoulos D: The study of HOX gene We thank the Seidel Family Trust and Dr. Jules Harris for function in hematopoietic, breast and lung carcinogenesis. their support. Anticancer Res 23: 2101-2110, 2003. 19* Burglin TR: A comprehensive classification of homeobox genes. References In: Guidebook to the Homeobox Genes. Duboule D (ed.). Oxford U Press, Oxford, pp. 25-71, 1994. http://homeobox.biosci.ki.se/ or References for which we could only obtain abstracts are www.homeobox.cjb.net/ maintained by Dr. Burglin. indicated with an asterisk. 20* Burglin TR: Homeodomain proteins, in Meyers RA (ed). 1 Chang HY, Chi J-T, Dudoit S et al: Diversity, topographic Encyclopedia of Molecular Cell Biology and Molecular differentiation, and positional memory in human fibroblasts. Medicine. Wiley-VCH Verlay GmbH @ Co. Weinheim, Proc Natl Acad Sci USA 99: 12877-12882, 2002. Germany pp. 179-222, 2005. 2 Rinn JL, Bondre C, Gladstone HB et al: (2006) Anatomic 21a Cillo C: Deregulation of the HOX Gene Network and Cancer, demarcation by positional variation in fibroblast gene in HOX Gene Expression. Papageorgiou S (ed.). pp. 121-133, expression programs. PLOS Genet 2(7): e119. doi:10.1371/ 2007. journal.pgen.0020119 21b Cillo C: HOX genes in human cancers. Invas Mets 14: 38-49, 3 Chanas G and Maschat F: Tissue specificity of hedgehog 1994. repression by the Polycomb group during 22a Spirov N, Bowler T and Reinitz J: Hox-Pro: A specialized melanogaster development. Mech Devel 122: 975-987, 2005. database for clusters and networks of homeobox genes. Nuc Acid 4 Ch’ng QL, Williams L, Lie YS et al: Identification of genes that Res 2: 337-340, 2000. regulate left-right asymmetric neuronal migration in 22b Spirov AV, Bopovsky M and Spirova OA: Pro DB: The . Genetics 164: 1355-1367, 2003. functional of HOX ensembles. Nuc Acid Res 30: 351- 5 Gillbert SG: , Sunderland MA (ed.). 353, 2002. hhtp://www.ephyb.nw.rv/labs38/ spirov/hox_pro/hox- 8th ed. Sinauer Assoc., pp. 347-348, 364-366, 2006. pro00.html 6 Sekimoto T, Yoshinobu K, Yoshida M et al: Region-specific 23a Banerjee-Basu S, Moreland TM, Hsu BJ et al: The expression of murine Hox genes implies the Hox code- homeodomain resource: 2003 update. Nuc Acid Res 31: 304-306, mediated patterning of the digestive tract. Genes Cells 3: 51- 2003. 64, 1998. 23b Banerjee-Basu S, Ryan JF and Baxevanis AD: The homeodomain 7 Golpon HA, Geraci MW, Moore MD et al: HOX genes in resource: a prototype database for a large protein family. Nucleic human lung. Altered expression in primary pulmonary Acids Res 28: 329-330, 2002. and emphysema. Am J Path 158: 955-966, 24 Chen K-N, Gu Z-D, Ke Y et al: Expression of 11 HOX genes 2001. is deregulated in esophageal squamous cell carcinoma. Clin 8 Horton SJ, Grier DG, McGonigle GJ et al: Continuous MLL- Cancer Res 11: 1044-1049, 2005. ENL expression is necessary to establish a "Hox code" and 25 Miller GJ, Miller HL, van Bokhoven A et al: Aberrant HOXC maintain immortalization of hematopoietic progenitor cells. expression accompanies the malignant phenotype in human Can Res 65: 9245-9252, 2005. prostate. Cancer Res 63: 5879-5888, 2003.

2150 Anderson et al: Homeobox-related “Positional” Genomic Information and Metastasis (Review)

26 Makiyama K, Hamada J, Takada M et al: Aberrant expression 44 Nottage MK, Kopciuk KA, Tzontcheva A et al: Analysis of of HOX genes in human invasive breast carcinoma. Oncol Rep incidence and prognostic factors for ispilateral breast tumour 13: 673-679, 2005. recurrence and its impact on disease-specific survival of women 27 Zhang X, Zhu T, Chen Y et al: Human growth - with node-negative breast cancer: a prospective cohort study. regulated HOXA1 is a human mammary epithelial . J Breast Cancer Res 8: R44, 2006. Biol Chem 278: 7580-7590, 2003. 45 Pergolizzi S, Settineri N, Santacaterina A et al: Ipsilateral 28 Chen H, Chung S and Sukumar S: HOXA5-induced apoptosis supraclavicular lymph nodes metastases from breast cancer as in breast cancer cells is mediated by caspases 2 and 8. Mol Cell only site of disseminated disease. Chemotherapy alone vrs Biol 24: 924-935, 2004. induction chemotherapy to radical radiation therapy. Ann 29 Kawagoe H, Humphries RK, Blair A et al: Expression of HOX Oncol 12: 1091-1095, 2001. genes, HOX cofactors, and MLL in phenotypically and 46 Nakagawa T, Okumura N, Miyoshi K et al: Prognostic factors functionally defined subpopulations of leukemia and normal in patients with ipsilateral pulmonary metastases from non- human hematopoietic cells. Leukemia 13: 687-698, 1999. small cell lung cancer. Eur J Cardiothorac Surg 28: 635-639, 30* Grier DG, Thompson A, Kwasniewska A et al: The 2005. pathophysiology of HOX genes and their role in cancer. J 47 Lian JB, Stein GS, Stein JL and van Wijnen AJ: Osteocalcin Pathol 205: 154-171, 2005. gene promotor: Unlocking the secrets for regulation of osteoblast 31 Wilson CS, Davidson GS, Martin SB et al: Gene expression growth and differentiation. J Cell Biochem 72: 62-72, 1999. profiling of adult acute leukemia identifies novel biologic 48 Shi X, Bai S, Li L and Cao X: Hoxa-9 represses transforming clusters for risk assessment and outcome prediction. Blood growth factor beta-induced osteopontin gene transcription. J 108: 685-696, 2006. Biol Chem 276: 850-855, 2001. 32 Abramovich C, Pineault N, Ohta H and Humphries RK: HOX 49 Cillo C, Cantile M, Mortarini R et al: Interactions among genes: from leukemia to hematopoietic stem cell expansion. HOX genes, inflammatory cytokines and adhesion molecules Ann NY Acad Sci 1044: 109-116, 2005. in human metastatic melanomas. Proc Am Assoc Cancer Res 33 Lee JM, Dedhar S, Kalluri R and Thompson EW: The 37: 75, #520, 1996. epithelial-mesenchymal transition: new insights in signaling, 50* Robbins SE, Shu WP, Kirschenbaum A et al: Bone extracellular development and disease. J Biol Chem 172: 973-981, 2006. matrix induces homeobox proteins independent of androgens: 34 Weinberg RA: The Biology of Cancer. Garland Sci., Taylor possible mechanism for androgen-independent growth in human and Francis, NY, pp. 597-624, 2007. prostate cancer cells. Prostate 29: 362-370, 1996. 35 Tomita K, van Bokhoven A, van Leenders GJLH et al: 51 Tarin D: The fallacy of the epithelial mesenchymal transition Cadherin switching in human prostate cancer progression. in neoplasia. Cancer Res 65: 5996-6001, 2005. Cancer Res 60: 3650-3654, 2000. 52 Mareel M and Leroy A: Clinical, cellular, and molecular 36 Wu X, Chen H, Parker B et al: HOXB7, a homeodomain aspects of cancer invasion. Physiol Rev 83: 337-376, 2003. protein, is overexpressed in breast cancer and confers epithelial- 53 Rusciano D, Welch DR and Burger MM: Introduction. In: mesenchymal transition. Cancer Res 66: 9527-9534, 2006. Laboratory Techniques in Biochemistry and Molecular 37 Yoshida H, Broaddus R, Cheng W et al: Deregulation of the Biology, Vol. 29. Cancer Metastases: Experimental approaches. HOXA10 homeobox gene in endometrial carcinoma: Role in S Pillai, PC van der Vliet (eds.). Elsevier, Amsterdam, 2000; the epithelial-mesenchymal transition. Cancer Res 66: 889- pp. 1-9 and chapters following. 897, 2006. 54* Cairns RA, Khokha R and Hill RP: Molecular mechanisms of 38 Riss J, Khanna C, Koo S et al: Cancers as wounds that do not tumor invasion and metastasis – an integrated review. Curr heal: differences and similarities between regeneration/repair Mol Med 3: 659-671, 2003. and renal cell carcinoma. Cancer Res 66: 7216-7224, 2006. 55 Kuo T-H, Kubota T, Watanabe M et al: Liver colonization 39 Anderson KM, Tsui P, Guinan P and Rubenstein M: To competence governs colon cancer metastasis. PNAS US 92: what extent are the "sets" of biological properties expressed 12085-12089, 1995. by hypoxic cancer cells and by cancer "stem" cells equivalent, 56 Keleg S, Buchler P, Ludwig R et al: Invasion and metastasis in intersecting, disjoint or complementary? Some implications pancreatic cancer. Mol Cancer 2: 14. doi:10.1186/1476-4598-2- for the design of cancer therapies. Med Hypo 68: 166-168, 14, 2003. 2007. 57* Moore MA: The role of chemoattraction in cancer metastases. 40 Jechlinger M, Grunert S, Tamir IH et al: Expression profiling Bioassays 23: 674-676, 2001. of epithelial plasticity in tumor progression. Oncogene 22: 58 Taichman RS, Cooper C, Keller ET et al: Use of the stromal 7155-7169, 2003. cell-derived factor-1/CXCR4 pathway in prostate cancer 41 Zajckowski DA, Bartholdi MF, Gong Y et al: Identification of metastasis to bone. Cancer Res 62: 1832-1837, 2002. gene expression profiles that predict the aggressive behavior of 59 Mundy GR: Metastasis to bone: causes, consequences and breast cancer cells. Cancer Res 61: 5168-5178, 2001. therapeutic opportunities. Nat Rev Cancer 2: 584-593, 2002. 42 Larue L and Bellacosa A: Epithelial–mesenchymal transition 60 Li X, Nie S, Chang C et al: Smads oppose Hox transcriptional in development and cancer: role of phosphatidylinositol 3’ activities. Exptl Cell Res 312: 854-864, 2006. kinase/AKT pathways. Oncogene 24(50): 7443-7454, 2005. 61 Kaplan RN, Rafii S and Lyden D: Preparing the "soil": the 43 Farrell CM, Dorsam ST, Ohta H et al: Activation of stem-cell premetastatic niche. Cancer Res 66: 11089-11093, 2006. specific genes by HOXA9 and HOXA10 homeodomain 62 Shaked Y, Ciarrocchi A, Franco M et al: Therapy-induced proteins in CD 34+ human cord blood cells. Stem Cells 23: acute recruitment of circulating endothelial progenitor cells to 644-655, 2005. tumors. Science 313: 1785-1787, 2006.

2151 ANTICANCER RESEARCH 27: 2141-2154 (2007)

63 Welch DR, Steeg PS and Rinker-Schaeffer CW: Molecular or only a temporal association of cited observations exists, biology of breast cancer metastasis: Genes of human breast which papers are considered the most essential in a field cancer metastasis. Breast Cancer Res 2: 408-416, 2000. and is appropriate priority given. Many of these "elements" 64 Welch DR and Hunter KW: A new member of the growing may not maintain the "connectivity" of specific datum family of metastasis suppressors identified in prostate cancer. J required for an extended and continuously valid argument, Natl Cancer Inst 95: 839-841, 2003. although some of the intermediate "steps" might be 65 Steeg PS, Bevilacqua G, Kopper L et al: Evidence for a novel dispensable and not invalidate the overall contention. gene associated with low tumor metastatic potential J Natl 75 Abboud CN and Lichtman MA: Structure of the marrow and Cancer Inst 80: 200-204, 1988. the hematopoietic microenvironment. In: Williams 66 Yang J, Mani SA, Donaher JL et al: Twist, a master regulator Hematology, 7th ed. Lichtman MA et al., McGraw Hill, NY, of morphogenesis, plays an essential role in tumor metastasis. pp. 45-52, 2004. Cell 117: 927-939, 2004. 76 Jones FS, Prediger EA, Bittner DA, Robertis EMD and 67a Woelfle U, Cloos J, Sauter G et al: Molecular signature Edelman GM: Cell adhesion molecules as targets for Hox associated with bone marrow metastasis in human breast genes: Neural cell adhesion molecule promoter activity is cancer. Can Res 63: 5679-5684, 2003. modulated by cotransfection with Hox-2.5 and -2.4. Proc Natl 67b Welsh JB, Zarrinkar PP, Sapinoso LM et al: Analysis of gene Acad Sci USA 89: 2086-2090, 1992. expression profiles in normal and neoplastic ovarian tissue 77 Divita V, Barber G, Odartchenko P et al: Expression of samples identified candidate molecular markers of epithelial homeobox-containing genes in primary and metastatic ovarian cancer. Proc Natl Acad Sci USA 98: 1176-1181, 2001. colorerctal cancers. Eur J Can 6: 887-893, 1993. 67c Jazaeri AA, Awtrey CS, Gadisetti VR et al: Gene expression 78 Feldman G, Dhara S, Fendrich V et al: Blockade of hedgehog profiles associated with response to chemotherapy in epithelial signaling inhibits pancreatic cancer invasion and metastases: A ovarian cancers. Clin Cancer Res 11: 6300-6310, 2005. new paradigm for combination therapy in solid cancers. Cancer 68 Bianchini M, Levy E, Zucchini C et al: Comparative study of Res 67: 2187-2196, 2007. gene expression by cDNA microarray in human colorectal 79 Roberts DJ, Johnson RL, Burke AC et al: Sonic hedghog is an cancer tissues and normal mucosa. Int J Oncol 29: 83-94, 2006. endodermal signal inducing BMP-4 and HOX genes during 69 L’Esperance S, Popa I, Bachvarova M et al: Gene expression induction and regionaliztion of the chick hindgut. Development profiling of paired ovarian tumors obtained prior to and 121: 3163-3174, 1995. following adjuvant chemotherapy: Int J Oncol 29: 5-24, 2006. 80 Thayer SP, di Magliano MP, Heisler PN et al: Hedgehog is an 70 Ramaswamy S, Ross KN, Lander ES and Golub TR: A early and late mediator of pancreatic cancer tumorigenesis. molecular signature of metastasis in primary solid cancers. Nat Nature 425: 851-856, 2003. Gen 33: 49-54, 2003. 81 Leroy P, Berto F, Bourget I and Rossi B: Down-regulation of 71 Sjoblom T, Jones S, Wood L et al: The consensus coding Hox A7 is required for cell adhesion and migration on sequences of human breast and colorectal cancers. Science 314: fibronectin during early HL-60 monocytic differentiation. J 268-274, 2006. Leuko Biol 75: 680-688, 2004. 72 Stevens TA, Iacovoni JS, Edelman DB and Meech R: 82 Novak P, Jensen T, Oshiro MM et al: Epigenetic inactivation Identification of novel binding elements and gene targets for the of the HOXA gene cluster in breast cancer. Cancer Res 66: homeodomain protein BARX2. J Biol Chem 279: 14520-14530, 10664-10670, 2006. 2004. 83 Raman V, Martensen SA, Reisman D et al: Compromised 73a Galili N, Davis RJ, Fredericks WJ et al: Fusion of a fork head HOXA5 further can limit P53 expression in human breast domain gene to PAX3 in the solid tumour alveolar tumours. Nature 405: 974-978, 2000. rhabdomyosarcoma. Nat Genet 5: 230-235, 1993. 84 Care A, Silvani A, Meccia E et al: HOXB7 constitutively 73b Fu SW, Schwartz A, Stevenson H et al: Correlation of activates basic in melanomas. Mol Cell expression of BP1, a homeobox gene, with receptor Biol 16: 4842-4851, 1996. status in breast cancer. Breast Cancer Res 5: R82-R87, 2003. 85 Maeda K, Hamada J-I, Takahashi Y et al: Altered expression 74a Hui H and Perfetti R: Pancreatic duodenum homeobox-1 of HOX genes in human cutaneous malignant melanoma. Int J regulates pancreas development during embryogenesis and islet Can 114: 436-441, 2005. cell function in adulthood. Eur J Endo 146: 129-141, 2002. 86 Naora H, Yang YQ, Montz FJ et al: A serologically identified 74b Six Degrees of Separation. Wikipedia.com. 2007. One tumor antigen encoded by a homeobox gene promotes growth additional pitfall of the "information age" is a form of over- of ovarian epithelial cells. Proc Natl Acad Sci USA 98: 4060- interpretation in which almost any elements of a hypothesis 4065, 2001. can be related to supporting data through a sufficiently 87 Care A, Felicetti F, Meccia E et al: HOXB7, a key factor for extensive chain of observations. Arguments consistent with tumor-associated angiogenesis. Can Res 61: 6532-6539, 2001. many different ideas can be developed, based on data 88 Gorski DH and Walsh K: The role of homeobox genes in vascular selected from apparent similarities between different remodeling and angiogenesis. Circ Res 87: 865-872, 2000. species, experimental systems and normal or malignant cells 89 Myers C, Charboneau A, Cheung I et al: Sustained expression expressing diverse properties. This is especially true for of homeobox D10 inhibits angiogenesis. Am J Path 161: 2099- those of us lacking direct experience in these several fields, 2109, 2002. sampling literature without the experts’ knowledge of the 90 Rhoads K, Arderiu A, Charboneau A et al: A role for Hox A5 subject. Unknown is the applicability of each element in regulating angiogenesis and vascular patterning. Lymphat employed, whether any causal relationship can be inferred Res Biol 3: 240-252, 2005.

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91 Hamada J-I, Omatsu T, Okada F et al: Overexpression of 106 Lei H, Wang H, Juan AH and Ruddle FH: The identification homeobox gene HOXD3 induces coordinate expression of of Hoxc8 target genes. Proc Natl Acad Sci USA 102: 2420- metastasis-related genes in human lung. Int J Cancer 93: 2424, 2005. 516-525, 2001. 107 Kawabe T, Muslin AJ and Korsmeyer SJ: HOX11 interacts 92 MiyazakiYJ, Hamada J-I, Tada M et al: HOXD3 enhances with protein phosphatases PP2A and PP1 and disrupts a G2/M motility and invasiveness through the TGF-B-dependent and - cell-cycle checkpoint. Nature 385: 454-458, 1997. independent pathways in A549 cells. Oncogene 21: 798-808, 2001. 108 Mallo GV, Soubeyran P, Lissitzky J-C et al: Expression of the 93 Okubo Y, Hamada J, Takahashi Y et al: Transduction of Cdx1 and Cdx2 homeotic genes leads to reduced malignancy in HOXD3-antisense into human melanoma cells results in colon cancer-derived cells. J Biol Chem 273: 14030-14036, 1998. decreased invasive and motile activities. Clin Exp Mets 19: 503- 109 Dequeant M-L, Glynn E, Gaudenz K et al: A complex 511, 2002. oscillating network of signaling genes underlies the mouse 94 Carrio M, Arderiu G, Myers C and Boudreau NJ: Homeobox segmentation . Science 314: 1595-1598, 2006. D10 induces phenotypic reversion of breast tumor cells in a 110 Anichini A, Mortarino R, Alberti S et al: T cell-receptor three-dimensional culture model. Can Res 65: 7177-7185, 2005. engagement and tumor ICAM-1 upregulation are required to 95 Waitregny D, Alami Y, Clausse N et al: Overexpression of the by-pass low susceptibility of melanoma cells to autologous homeobox gene HOXC8 in human prostate cancer correlates CTL-mediated lysis. Int J Cancer 53: 305-329, 2002. with loss of tumor differentiation. The Prostate 50: 162-165, 2002. 111 Green SK, Karlsson MCI, Ravetch JV and Kerbel RS: 96 Jung C, Kim R-S, Zhang H-J et al: HOXB13 induces growth Disruption of cell-cell adhesion enhances antibody-dependent suppression of prostate cancer cells as a repressor of cellular cytotoxicity: Implications for antibody-based therapies hormone-activated signaling. Cancer Res of cancer. Cancer Res 62: 6891-6900, 2002. 64: 9185-9192, 2004. 112* Trikha M, Zhou Z, Nemeth JA et al: CNTO 95, a fully 97 Daftary GS and Taylor HS: Endocrine regulation of HOX human monoclonal antibody that inhibits av integrins, has genes. Endo Rev 27: 331-355, 2006. antitumor and antiangiogenic activity in vivo. Int J Cancer 110: 98a Edelman GM and Jones FS: Gene regulation of cell adhesion 326-335, 2004. molecules in neural morphogenesis. Acta Paed Suppl 422: 1-9, 113 Li B, Lalani AS, Harding TC et al: Vascular endothelial growth 1997. factor blockade reduces intratumoral regulatory T cells and 98b Edelman GM and Jones FS: Outside and downstream of the enhances the of a GM-CSF-secreting cancer homeobox. J Biol Chem 268: 20683-20686, 1993. immunotherapy. Clin Cancer Res 12: 6808-6816, 2006. 99 Cillo C, Cantile M, Mortarini R et al: Differential patterns of 114 Wicha MS, Liu S and Dontu G: Cancer stem cells: an old idea- HOX gene expression are associated with specific integrin and A paradigm shift. Cancer Res 66: 1883-1890, 2006. ICAM profiles in clonal populations isolated from a single 115 Kung AL, Wang S, Klco JM et al: Suppression of tumor growth human melanoma metastasis. Int J Cancer 66: 692-697, 1996. through disruption of hypoxia-inducible transcription. Nat Med 100 Gress TM, Muller-Pillasch F, Lerch MM et al: Expression and 6: 1335-1340, 2006. in situ localization of genes coding for extracellular matrix proteins and extracellular matrix degrading proteins and Note in press: In a study in Nature (April 12, 2007), Massague and extracellular matrix degrading proteases in pancreatic cancer. colleagues identified 4 genes, coding for proteins epiregulin, Int J Cancer 62: 407-413, 1995. COX2, and matrix metalloproteinases 1 and 2 which when silenced 101 Fiucci G, Ravid D, Reich R and Liscovitch M: Caveolin-1 individually, limited the growth of human breast cancers metastatic inhibits anchorage-independent growth, anoikis and to the lungs in mice. Reducing the function of all four invasiveness in MCF-7 human breast cancer cells. Oncogene simultaneously almost completely eliminated tumor growth by 21: 2365-2375, 2002. limiting their blood supply. In an article in Proc Natl Acad Sci USA 102 Bruhl T, Urbich C, Aicher D et al: Homeobox A9 on line (April 9, 2007), Massague and colleagues examined 18 transcriptionally regulates the EphB4 receptor to modulate genes denoted as constituting a “lung metastasis gene expression endothelial cell migration and tube formation. Circ Res 94: signature”, the LMS designation. Breast cancers among 738 743-751, 2004. analyzed exhibiting an active LMS were associated with metastases 103 Chen J and Ruley HE: An enhancer element in the EphA2 to lungs and with active vascularization. (Eck) gene sufficient for rhombomere-specific expression is activated by HOXA1 and HOXB1 homeobox proteins. J Biol Chem 273: 24670-24675, 1998. 104 Knosp WM, Scott V, Bachinger HP and Stadler HS: HOXA13 regulates the expression of bone morphogenetic proteins 2 and 7 to control distal limb morphogenesis. Develop 131: 4581- 4592, 2004. 105 Dai J, Keller J, Zhang J et al: Bone morphogenetic protein-6 promotes osteoblastic prostate cancer bone metastases through Received April 4, 2007 a dual mechanism. Cancer Res 65: 8274-8285, 2005. Accepted April 18, 2007

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