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Vol. 4, 567-576, March 1998 Clinical Cancer Research 567

The Overexpression of RHAMM, a Hyaluronan-binding Protein

That Regulates ras Signaling, Correlates with Overexpression

of Mitogen-activated Protein Kinase and Is a Significant

Parameter in Breast Cancer Progression’

Chao Wang, Ann D. Thor, Dan H. Moore II,

Yong Zhao, Russell Kerschmann, Robert Stern,

Peter H. Watson, and Eva A. Turley2

Department of Anatomy and Cell Biology, the University of Toronto.

and The Hospital for Sick Children, Toronto, Ontario MSG lX8,

Canada [C. W., Y. Z., E. A. T.J: Department of Pathology andPhysiology, the University of Manitoba, Winnipeg, Manitoba

R3E 0W3, Canada [P. H. W.]; Department of Pathology. theUniversity of California at San Francisco, San Francisco, California

94143 [R. K., R. 5]; Department of Pathology, Evanston Hospital

and Northwestern University, Evanston, Illinois 60201 [A. D. 1.]; andCalifornia Pacific Medical Center, San Francisco, California 941 10

[D.H.M.]

ABSTRACT

RHAMM is an oncogene that regulates signaling

through ras and controls mitogen-activated protein kinase[extracellular signal-regulated protein kinase (ERK)] ex-pression in embryonic murine fibroblasts. ERK is a dual-

specificity kinase that controls expression of proteins rele-vant to tumorigenesis, proliferation, and motility. To assesswhether RHAMM and ERK are involved in human breasttumor progression, we examined RHAMM, ras, and ERKexpression in two cohorts of breast cancer patients using

reverse transcription-PCR and immunocytochemistry. We

show that overexpression of RHAMM in primary tumors of

two patient cohorts was significantly prognostic of poor

outcome in breast cancer progression. Furthermore,RHAMM overexpression occurred within subsets of tumor

cells in the primary tumor, and this staining pattern wasassociated with lymph node metastases. The metastases ex-hibited a significantly higher level of staining for RHAMM

Received 9/22/97; revised 12/12/97; accepted 12/16/97.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

1 This study was supported by the National Cancer Institute of Canada

(NCIC) Terry Fox Award and Hyal Pharmaceutical Co., Mississauga,Canada (to E. A. T.), NIH Grant P0-CA 44768 (to R. S., D. M., andA. D. T.), RO GMS (to R. S.), the Canadian Breast Cancer ResearchInitiative (to P. H. W.), a Manitoba Health Research Council studentship(to C. W.), and a Children Hospital Research Foundation scholarship(to E. A. T.). The Manitoba Breast Tumor Bank is supported by theNCIC.2 To whom requests for reprints should be addressed. at The Hospital forSick Children, 555 University Avenue, Toronto, Ontario M5G !X8,

Canada. Phone: (416)813-8201; Fax: (416)813-7480: E-mail:[email protected].

than did the primary tumor. RHAMM expression strongly

correlated with overexpression of both ras and ERK, al-

though overexpression of either of these two signaling mel-

ecules was not by itself a prognostic indicator. These results

identify a new parameter that is involved in lymph node

metastasis of primary breast cancers and suggest that quan-tification of RHAMM overexpression may be a useful prog-

nostic indicator for breast carcinoma progression.

INTRODUCTION

Breast cancer is a complex and heterogeneous disease that

is the most common worldwide cause of malignancy in women

(1). Familial breast cancer involves the BRCA I and BRCA2

genes (2, 3), but the underlying molecular basis of the most

common, nonhereditary forms of breast cancer are not yet

clearly defined, although a variety of relatively crude parameters

have been linked to prognosis of this disease, including tumor

size and presence of lymph node metastasis ( 1). ER3 status has

also long been linked to outcome of nonfamilial breast cancer.

More recently, oncogenes, such as c-erbB-2, and tumor suppres-

sor genes, such as p53, have been proven not only to be useful

prognostic parameters but also to provide a first step toward

defining the molecular basis of this disease ( 1 , 4, 5). The

complex etiology of nonfamilial breast cancer suggests, how-

ever, that multiple molecular parameters are involved in pro-

gression of this disease.

It is interesting that mutation of the oncogene p21 ras has

been implicated in many human tumors. including pancreatic,

lung, and colorectal malignancies (6, 7), but this GTP-binding

protein does not appear to play a major role in human breast

carcinoma (5, 6). However, overexpression and activation of a

downstream kinase, the MAP kinase ERK, has recently been

linked to lymph node metastasis of breast tumors (8), raising the

interesting possibility that deregulation of elements downstream

of ras may play a role in tumor progression in the absence of

activation of ras by mutation. The molecular mechanism(s)

responsible for hyper-activation/expression of MAP kinase is

not yet understood.

We have begun to investigate the involvement of proteins

that regulate the ras-ERK pathway in human tumor progression.

For several reasons, we have investigated the relationship of the

HA-binding protein RHAMM to breast cancer progression.

3 The abbreviations used are: ER, estrogen receptor; HA, hyaluronan;

ERK, extracellu!ar signal-regulated protein kinase; MAP, mitogen-ac-tivated protein; RHAMM, receptor for hyaluronan-mediated motility;

RT. reverse transcription; TBS, Iris-buffered saline.

Research. on September 10, 2021. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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4 5. Mohapatra. D. Dubik. S. Zhang. E. A. Turley, and A. H. Greenberg.

submitted for publication.

568 RHAMM and Breast Cancer

Firstly. RHAMM is an oncogene when overexpressed in murine

fibroblasts and is functionally linked to ras signaling (9). Sec-

ondly. RHAMM controls expression levels of ERK and its

upstream activator MAPK/ERK kinase in vitro.4 Finally,

RHAMM has recently been shown to be overexpressed in

malignant pancreatic cells and lung carcinoma cells that have

deregulated ras signaling (10. 1 1). In this study. we have eval-

uated the prognostic value of RHAMM, ras, and ERK overex-

pression in breast cancer and the relationship of RHAMM to ras

and ERK overexpression in this disease.

PATIENTS AND METHODS

Patients and Samples. The first cohort consisted of 98

human breast tumor specimens obtained from the National

Cancer Institute of Canada-Manitoba Breast Tumor Bank. In all

cases, specimens obtained from the bank had been snap frozen

at -70#{176}Cafter surgical removal. Tumors were selected from the

Tumor Bank database to represent a range of pathological

grades (Nottingham system, score 4-9, corresponding to low to

high grade; Ref. 12) and ER status (as determined by a ligand

binding assay). Specific frozen tissue blocks were chosen in

each case on the basis of several further criteria. These criteria

included a cellular content of greater than 30% invasive tumor

cells with minimal normal lobular or ductal epithelial compo-

nents, good histological preservation. and absence of necrosis.

The majority of tumors were primary invasive ductal carcino-

mas. This cohort was used to determine the relationship of

RHAMM mRNA expression with previously determined patho-

biological factors.

The second cohort comprised archival materials from pri-

mary invasive breast carcinomas of 400 patients that had been

surgically excised at the Massachusetts General Hospital from

1979 to 1982. These were used to determine the relationship of

RHAMM protein overexpression with previously determined

pathobiological factors and with survival. These patients con-

tinued their clinical care at Massachusetts General Hospital. The

following information was obtained from the patients’ clinical

and medical records: age at diagnosis, location of primary

tumor, time to metastasis, site of metastasis, therapeutic inter-

vention, overall survival time, and cause of death. The median

follow-up time was 10.6 years. with a minimum of 1 year and a

maximum of I 6 years: 75% of cases had follow-up of greater

than 10 years.

Antibodies. The polyclonal antibody used in this study,

R3, was raised in rabbits to a specific peptide (amino acids

269-288: Ref. 13) encoded in the murine RHAMM cDNA,

which is conserved in human RHAMM cDNA (14). Rabbit IgG

and R3 preincubated with murine RHAMM fusion protein were

used as control. Monoclonal antibody to p21 ras (ras-lO) was

purchased from Oncogene Science (Manasset, NY).

Extraction of RNA. Total RNA was extracted from one

to three 20-p.m frozen tumor sections, as described previously

( 15), using a small-scale RNA extraction protocol (Tri-Reagent,

Molecular Research Center, Inc., Cincinnati, OH), ensuring a

direct correlation between the material analyzed and histologi-

cally assessed cellular composition. The yield from tumor sec-

tions was quantified by a spectrophotometer in a 50-�il micro-

cuvette. The average yield of total RNA per 20 p.m section was

4 p.g/cm2 (±20% variation with cellularity), and this was asso-

ciated with a consistent A2M),,,,o > 1.8.

RT-PCR Analysis. The expression of RHAMM and

ERK was assessed by RT-PCR, followed by agarose electro-

phoresis and ethidium bromide staining to visualize the PCR

products. Amplification of actin was performed in parallel to

control for reliability of RT of amplification. RHAMM isoform

bands and ERK bands were then assessed by subjective scoring

ofband presence and intensity (-, ±, +, ++, or +++).

RT was performed with 100 ng of total RNA with 1 mr�i

dNTP, 1 unit of RNase inhibitor, 2.5 m�t oligodeoxythymidylic

acid primer, 50 units of Moloney murine leukemia virus reverse

transcriptase and 1 X Moloney murine leukemia virus buffer

(Life Technologies, Inc.) in a total volume of 10 �i.l of6O mm at

37#{176}C.Following S mm of incubation at 95#{176}C,the reaction was

then diluted to 40 iil, and 1 pi of the cDNA (equivalent to 2.5

ng of the input RNA) was then subjected to PCR.

PCR amplifications were conducted separately for

RHAMM and ERK using I p.1 of RT mixture in a volume of 50

p.1, in the presence of 10 mM Tris-HC1 (pH 8.3), 50 mrsi HC1, 1.5

mM MgC12, 0.2 nmi dATP, 0.2 mist dCTP, 0.2 mM dGTP, 0.2 mtvi

dTTP, 100 ng of each primer, and 2 units of Taq DNA polym-

erase. The primers used for RHAMM were as follows: sense

orientation, GCAAACACTGGATGAGCTfGA; antisense on-

entation, TGGTCTGCTGATCTAGAAGCA. The primers for

ERK were as follows: sense orientation, GCAGGTGT1’C-

GACGTGGG; antisense orientation, GTGCAGAACGT-

TAGCTGAAT. PCR cycling parameters were denaturation at

94#{176}Cfor 4 mm, denaturation at 94#{176}Cfor 45 s, annealing at 60#{176}C

for RHAMM and 56#{176}Cfor ERK for 45 s, and extension at 72#{176}C

for 2 mm. Forty-five cycles were used with a final extension

time of 8 mm. RT-PCR products were analyzed on 1 % agarose

gels with ethidium bromide (200 ng/ml). For RHAMM, the

4l6-bp band and, in some cases, an additional 266-bp band were

observed. These bands were cut out for sequencing. For ERK, a

band of 393 bp was observed. Semiquantitative analysis of the

relative amounts of RHAMM or ERK transcripts expressed was

performed by comparing the expression of the RHAMM or ERK

gene with that of human actin gene, the primers for which were

as follows: sense orientation, ATCTGGCACCACACC1TCTA-

CAATGAGCTGCG; antisense orientation, CGTCTACAC-

CTAGTCG1TCGTCCTCATACTGC. This resulted in an

838-bp fragment (Clontech).

DNA Sequencing. The DNA excised from the ethidium

bromide stained agarose gel was purified using a Prep-A-Gene

DNA purification system (Bio-Rad) according to the manufac-

turer’s instructions and cloned into the pCR TA vector (Invitro-

gen, San Diego, CA). It was then sequenced by the dideoxy

chain termination method using the T7 Sequencing kit (Phar-

macia Biotech, Uppsala, Sweden). A 4l6-bp insert corre-

sponded to part of the previously reported human RHAMM

isoform [designated RHAMM (+E9)], whereas a 266-bp insert

corresponded to RHAMM minus exon 9 sequence [designated

RHAMM (-E9)] (14, 16).



Clinical Cancer Research 569

Immunohistochemistry. Routine formalin-fixed, par-

affin-embedded tissues were cut into 4-rim sections and

mounted on polylysine-coated slides for assessing RHAMM

and ras expression. The avidin-biotin-peroxidase complex

method was used, as described previously for CD44 staining

(17), but with the following modifications. The slides were

incubated with 1.5% goat serum in 0.01 M TBS for 1 h to

block nonspecific binding. The primary antibody (13, 18)

was raised in rabbits to a peptide as positions 269-288,

encoded in the murine RHAMM cDNA (13) and conserved in

human RHAMM cDNA (14). This was diluted with 1.5% goat

serum/TBS (1 :600) and incubated on slides overnight at 4#{176}C.

ras staining was performed using the ras-lO antibody at

1 :2000 in PBS with 1% BSA (Oncogene Science, Manasset,

NY). Endogenous peroxidase activity was blocked by incu-

bating the slides with 0.6% H202 in methanol (Mallinckrodt)

for 30 mm at room temperature. The dilution of antibody was

chosen by determining the dilution at which no staining for

reduction mammoplasties was observed. The slides were then

incubated with biotinylated goat antirabbit IgG [Vectastain

ABC peroxidase kit, Vector Laboratories, Inc. (Burlingame,

CA), 1:200 in 0.01 M TBS] for 1 h at room temperature,

followed by an avidin-biotin-peroxidase complex (Vec-

tastain, Vector Laboratories, Inc., 1:200 in 0.01 M TBS) to

visualize bound antibody. Between each step, the slides were

washed three times with 0.01 M TBS. The peroxidase activity

was developed by incubation in 0.05% 3,3’-diaminobenzi-

dine (Sigma Chemical Co.) and 0.1% H202 in 0.05 M TBS.

The slides were counterstained with methyl green. Nonim-

mune sera as well as antibody preabsorbed with RHAMM

fusion (recombinant) protein, was used as a negative control.

The extent of reactivity of human breast cancer tissues to

RHAMM was assessed by two independent, blinded observers

[C. W. (University of Toronto) and R. S. (University of Cali-

fornia at San Francisco)] without knowledge of clinical out-

come. The staining intensity was scored using an arbitrary scale

of 0-4+ (0, negative; 4+, strongly positive). ras staining was

scored by R. K. (at University of California at San Francisco)

using a 0-3 + system (0, negative; 3 + , strongly positive).

Scores were blinded as to patient outcome and other tumor

characteristics.

Four measures of staining intensity were tested. It was not

known a priori which of the four scoring measures would turn

out to be significant nor what cut-point would be useful for any

of them. These four measures were as follows: (a) general

overall intensity of staining; (b) scoring of isolated single or

multiple individual cells containing the most intense staining,

referred to as “focal staining”; (c) staining within peritumor

stroma; and (tO nuclear staining. The impetus for scoring focal

staining came from the custom in surgical pathology to infer the

overall diagnostic evaluation of a malignancy from the “worst”

or most ominous area of a slide.

Statistical Methods. Kaplan-Meier survival curves were

plotted for each of the scoring measures of immunocytochem-

istry (19). Differences between survival curves were generated

by using a cut-point to divide each scoring measure into a

dichotomous rating (0 if below and 1 if above the cut-point) and

were tested by the log rank (Mantel-Cox) test (20). A Cox

proportional hazard model was used to determine the signifi-

Table 1 RHAMM mRNA exp ression and tumor grade

RHAMM isoform Case No. Median grade P”

RHAMM (+E9)�+ 44 6�++ 54 7 0.0466

RHAMM (-E9)�± 70 7

� + 28 8 0.0163

RHAMM total�+ 40 6� ++ 54 7 0.0357

a Mann-Whimey u test.

cance of multiple factors in predicting survival. We used the

Survival Tools for StatView program to perform the statistical

analyses (19). To enhance assessment of the relationship of

focal staining alone, in the absence of contamination from

general staining, values for general staining were subtracted

from focal staining and reanalyzed. GRAPHPAD Prism soft-

ware was used to test the difference of RHAMM and ERK

mRNA expression in RT-PCR analyses.

RESULTS

RT-PCR Analysis of RHAMM mRNA in Primary

Breast Carcinoma Shows Prognostic Relationship to TumorProgression. To assess whether RHAMM expression is a

prognostic parameter in human breast cancer, we first investi-

gated RHAMM expression in a Manitoba patient cohort (n =

98) using RT-PCR of mRNA extracted from one to three con-

secutive 20-p.m frozen tissue sections. These cases were se-

lected to provide a range of tumor grade and ER/progesterone

receptor status.

Two RT-PCR products were obtained using primers from

exons 7 and 10 of the RHAMM gene (14, 16). One was repre-

sented as a cDNA insert of 416-bp [RHAMM (+E9); Refs. 14

and 16], and this form was the most common, occurring in all

tumors. Another isoform was represented as a cDNA insert of

266 bp that contained a deletion of exon 9 [RHAMM (-E9);

Refs. 14 and 16] and occurred in 29% of tumors. Elevated

expression of both RHAMM isoforms (i.e. , � + compared to �

+ +) showed a significant association with higher tumor grade

(P = 0.0466, 0.0163, and 0.0357, respectively; Table 1). Further

analysis of subsets of patients exhibiting other combined param-

eters of poor prognosis (high grade, ER negativity, and lymph

node positivity; n = 12) versus patients with good prognostic

parameters (low grade, ER positivity, and lymph node negativ-

ity; n = 15) showed a similar significant association of

RHAMM expression with poor prognosis (P 0.0063, 0.0085,

and 0.0213, respectively; Table 2). RHAMM (-E9) splicing

pattern did not differ in its prognostic value from the major

RHAMM isoform [RHAMM (+E9)] detected in these samples,

and the significance of RHAMM (-E9) is therefore not clear.

Protein translated from the RHAMM (-E9) isoform would not

be recognized by the antibody used for the immunohistochem-

ical analysis (see below), because this is directed to an exon 9

epitope (13, 16).

Immunostaining for RHAMM Is Heterogeneous. Weexamined RHAMM cellular distribution and expression level in




Table 2 RHAMM mRNA expression a nd prognostic parameters”

RHAMM isoformPoor prognosis

(12 cases)Good prognosis

(15 cases) P”

RHAMM (+E9)� +

� ++

RHAMM (-E9)� ±

� +

RHAMM total� +

� ++

2(17%)

10(83%)

5(42%)

7 (58%)

3 (25%)

9(75%)

11(73%)

4(27%)

14(93%)

1 (7%)

1 1 (73%)

4(27%)

0.0063

0.0085

0.0213

“ Poor prognosis parameters: high grade/ER-/node+. Good prog-

nosis parameters: low grade/ER +/node -.

,, Fisher’s exact test.

breast carcinoma tissue of a second patient cohort by immuno-

histochemistry (n = 400). Several RHAMM staining patterns

were observed. Widely present staining for the RHAMM

protein. termed “general staining,” was highly variable, rang-

ing from most cells being negative (-) to most cells being

very strongly positive (4+; Table 3; Fig. 1, A-C, arrow-

heads, and see Fig. 1 , B-E for variability). General staining

was observed both within tumor cells (Fig. 1, A-D) and, in

fewer cases, in the extracellular milieu, i.e. , the stroma sur-

rounding the tumor (Fig. lE and Table 3), consistent with

previous reports of the occurrence of intracellular and soluble

forms of RHAMM in murine cells (13, 21). Intracellular

RHAMM appeared to be both cytoplasmic and, in some

instances, nuclear (Fig. 1D). Again, this is consistent with the

presence of RHAMM in the cytoplasm and nucleus of the

cultured murine cells (13) and human tumor cells (11). Most

importantly, in some tumors, RHAMM was noticeably over-

expressed in small groups or individual cells within the

primary tumor (Fig. 1, B and C, arrows). Staining of these

cells was defined as focal staining.

RHAMM Focal Overexpression Is of Independent

Prognostic Value in Human Breast Cancer. Univariate

analyses of breast carcinoma tissue sections of the Boston

patient cohort found lymph nodal status (P < 0.0001) and tumor

size (P = 0.03) to be statistically significant for predicting

metastasis-free and overall survival (Table 4). RHAMM over-

expression in foci was significantly associated with poor prog-

nosis (P = 0.03) as an independent factor (Tables 4 and 5). A

high level of general staining was not significant (Table 4).

These results suggest that the focal expression of RHAMM

likely contributed to the results obtained in the first study.

To further assess the relationship of focal staining to breast

cancer metastasis, focal staining minus general background

staining (focal-general) was analyzed with respect to lymph

node-positive and lymph node-negative patients and other

standard factors (outlined in Table 4) with a Cox proportional

hazard model (19, 20). When the modified data were segregated

according to lymph node status, the focal-general parameter for

RHAMM allowed a separation of survival curves in both lymph

node-positive and lymph node-negative groups into subgroups,

and the difference between the groups was significant at P =

0.008 for overall survival and significant at P = 0.016 for

metastasis-free survival (Fig. 2; Table 5), suggesting that the

Table 3 Distribution of staining scores among 400 breast tumors

Staining Stromal Nuclear General tumor Focal tumor

0.0 331 323 21 3

0.5 35 16 74 24

1.0 18 10 73 28

1.5 8 5 92 54

2.0 3 18 86 69

2.5 4 2 34 70

3.0 1 14 17 723.5 0 4 3 54

4.0 0 8 0 26

presence of RHAMM focal staining parameter enhances prog-

nostic value of lymph node status. Interestingly, the odds ratios

for the modified focal staining parameter in Table 5 suggest that

when the difference between focal and background general

staining (i.e., focal staining minus general staining) is � 1 unit,

the chance of recurrence of metastasis is 1 .40 times as high as

when the staining difference is < 1 unit. Similarly, the chance of

death is 1.59 times as high for those tumors when the difference

is � 1 compared to when the difference is < 1 unit (also see Fig.

2). These results suggest that the appearance of focal staining of

highly RHAMM-positive cells against a low background of

general RHAMM staining are of particular significance to breast

cancer progression. The significance of this finding is not clear

at present.

To further assess the significance of high overexpression

of RHAMM to tumor progression in the second patient

cohort, tissues containing breast cancer metastases from 34

patients from the original patient cohort above were ran-

domly selected and were stained for RHAMM. The average

staining unit was compared to the average staining unit that

occurred within primary tumors. As shown in Table 6, me-

tastases exhibited a significantly higher score for RHAMM

staining than the primary tumors (P < 0.001). Furthermore,

95% of metastasis arose from primary tumors that contained

focal staining of �3.0. This result substantiates a relationship

between focal staining for RHAMM within the primary tu-

mor and the appearance of lymph node metastases, raising the

possibility that the foci in primary tumors contribute the

cancer cells in the lymph nodes. Alternatively, the high

expression of RHAMM may simply confer a growth advan-

tage to breast cancer cells within lymph nodes, and such cells

are secondarily selected for.

RHAMM Overexpression Correlates with Both ms andERK Overexpression, but ras and ERK Overexpression AreNot by Themselves Prognostic Parameters. Although only

a small percentage (approximately 5%) of breast cancers

exhibit ras mutations, an estimated 70% of breast cancers

overexpress this GTP-binding protein (22, 23). However, the

prognostic value of this occurrence is controversial ( 1 , 5, 23,

24). We found that ras overexpression by itself did not act as

a significant prognostic indicator (data not shown). Because

we have previously shown that ras regulates RHAMM ex-

pression (13), we also assessed whether ras expression and

RHAMM expression were correlated. No attempt was made

to detect mutations in ras. As shown in Table 7, expression

of general staining for RHAMM and ras was significantly



�1. �

‘el.

% &Ie�Stt� �‘�

p�.�,�‘ �..

..� %-.

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D

Fig. I Immunohistochemical staining of formalin-fixed, paraffin-embedded human breast cancer tissues using an antibody to RHAMM. Sectionswere counterstained with methyl green. The staining intensity of tumor cells and stroma is variable. A-C, general tumor staining (arrowheads) with

tumor focal staining in individual cells (arrows). D, tumor cells and stroma, both staining positively for RHAMM. E. tumors showing positive nuclearand cytoplasmic staining. F. tumors showing negative staining. A and F, X400; B, D, and E, X250; C, X650.

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correlated (r = 0.27; P = 0.038). However, interestingly, a

relationship between focal RHAMM staining and ras expres-

sion was not detected.

It has recently been demonstrated that overexpression

and activation of the dual-specificity MAP kinase ERK that

acts downstream of ras occurs within malignant cells at the

primary tumor site and in lymph node metastases of breast

cancer but not in benign lesions (8). We noted here that ERK

overexpression detected by RT-PCR in primary tumors of the

first cohort patients is not by itself significantly associated

with poor prognosis, although a clear trend exists between

increasing ERK levels and increasing tumor grade. as well as

between increasing ERK levels and positive node status (data

not shown). Because we have recently shown that HA/recep-

tor interaction regulates the expression of MAP kinases,4 we

assessed whether or not RHAMM overexpression correlated

with ERK overexpression. As shown in Table 8, RHAMM

overexpression strongly correlated with ERK overexpression

(r = 0.38; P < 0.0001).

Although we have noted that RHAMM levels (comparing �




Table 4 Univariate analysis of prognostic indicators for metastasis-free and overall survival

Metastasis-free survival Overall survival

S-yr survival 5-yr survival

Factor No. (%) P No. (%) P

Nodal status

None positive 179 83 186 80

> I positive 62 50 <0.0001 179 60 <0.0001

Tumor size (cm)2 181 70 199 72

2.01-5 149 69 160 69

>5 22 40 0.03 26 58 0.01

Tumor gradelor2 292 70 235 72

3 71 62 0.74 155 64 0.53

ER status

Negative 23 74 24 70

Positive 173 74 0.75 187 78 0.67

Age<50 96 65 103 68

�50 265 65 0.75 293 68 0.19

Stromal stainNone 301 70 329 70

Some 62 54 0.20 69 28 0.13

General stain0-0.5 85 70 95 72

1-1.5 152 58 165 62

>2 126 68 0.17 138 70 0.15

Focal stain0-1 46 83 55 78

1.5-2 113 62 123 65

2.5-3 132 62 141 66

3.5-4 71 68 0.03 78 70 0.07

Nuclear stain

None 292 72 322 68

Some 7 1 62 0.74 76 68 0.64

Table 5 Multivariate analysis of prognostic factors for metastasis-free and overall survival

Metastasis-free survival Overall survival

Factors Odds ratio P Odds ratio P

Nodal status 2.96” <0.0001 2.14a 0.0001

Focal-general RHAMM 1.40” <0.016 159b <0.008

Tumor size <0.012-5 cm 1 .25’ <0.02 1 .46c

>5 cm 1.99’ <0.003 1.61’

<0.004<0.08

<0.002U Odds ratio for � versus 0 positive nodes.

b Odds ratio for combined RHAMM staining � versus < I.

C Odds ratio for tumor size shown versus tumor size �2 cm.

+ to � + +) were prognostic of poor outcome in the first patient

cohort of98 cases (Tables 1 and 2), comparison oflevels within the

high RHAMM tumor subgroup did not show significant differ-

ences in terms of tumor grade (i.e. , + + versus + + +). However,

these subgroups could be sorted into different tumor grades if ERK

expression was also included. Thus, the subset oftumors in the high

RHAMM group that exhibited high RHAMM and high ERK

overexpression (+ + +, + + + ; 21 patients) were of a higher tumor

grade than those that expressed lower RHAMM/high ERK (+ +,

+ + +; 30 patients; P = 0.034; Table 9). A trend also existed

within subgroups of tumors. High RHAMM/low ERK subgroups

contained lower grade tumors than high RHAMM/high ERK sub-

groups (data not shown). These results suggest a relationship

among RHAMM and ERK expression and breast cancer progres-

sion. However, only a small number of patients were investigated

in this study; further work will be needed to establish this relation-

ship and to determine whether ratios of RHAMM/ERK provide an

independent prognostic variable.



1.0

0.9

0.8

c: 0.70

� 0.6

�0.5

.� 0.4

U) 0.3

0.2

0.1

0

General staining, average score

Focal staining, average score

C, Mann-Whitney U test.

A

B

0 2 4 6 8 10 12 14 16

Years since diagnosis

0.9

.� 0.8

U- 0.70)C

.� 06

U)

a)‘� 0.4

Cl)

.� 0.3

� 0.2

0.1

4 6 8 10

Years since diagnosis


Fig. 2 Kaplan-Meier survival curves for overall survival (A) and me-

tastasis-free survival (B). The top two curves in each plot show node-negative patients, and the bottom two curves indicate node-positivepatients. 0 and 0. tumors with focal-general staining parameter <1unit; #{149}and S, tumors with values � I unit. Numbers of patients in eachgroup are given in Table 4. The differences between survivals weretested by the log rank (Mantel-Cox) test.

DISCUSSION

It is now generally accepted that tumorigenesis is a multi-

ple-step process that results from cumulative genetic alterations

in proto-oncogenes and tumor suppressor genes, as well as DNA

repair genes (25, 26). This classic genetic model originally

described by Fearon and Vogelstein (7) in colorectal cancer now

serves as a paradigm for many malignancies. In this model, the

development of metastatic colorectal cancer from normal cob-

rectal epithelial cells involves alterations of at least four inde-

pendent genes, namely, APc, K-Ras, DCC, and p.53, and addi-

Table 6 RHAMM expression in primary and

RHAMM staining Primary tumor

metastatic tumors

Metastasis P”

1.4 2.8 <0.0012.8 3.6 <0.001

tional changes, such as altered DNA methylation, as well as

inactivation of MSH2 and MLHJ genes (25). Furthermore, al-

teration of other genes, such as TGF-�3 type II receptor, may also

be necessary to accelerate the process of coborectal cancer

progression (25). Although several genes, such as c-erbB-2,

p53. and urokinase, have been implicated in nonfamilial breast

cancer progression (4, 5), a similar molecular basis of this

disease has not been identified. Our results suggest that over-

expression of ERK and a factor that regulates ERK expression,

RHAMM, may play a role in early metastasis of breast cancer.

ERK is a protein kinase that is activated by dual phosphoryla-

tion on threonine and tyrosine residues. It is a key regulator of

cell behavior, because it controls cell proliferation and activa-

tion of transcription factor AP1 (which controls expression of

proteins relevant to tumor invasion; Refs. 27 and 28). Recently,

ERK has also been shown to control cell motility (29, 30), a

property thought to be a key to metastasis (3 1, 32). RHAMM is

a HA-binding protein that also controls cell proliferation and

motility (9, 33). It does not bear significant homology to other

classes of proteins and is therefore not a known enzyme, nor

does it bear protein motifs, such as SH2 and SH3-domains, that

are sites for other signaling molecules (13, 14, 16). Our results

suggest that although progression of a specific neoplasm may

not involve overexpression or activation of ras, downstream

effectors of this GTP-binding protein can be deregulated and

contribute significantly to cancer progression. Our results also

demonstrate that regulation of the ras-ERK kinase cascade is

complex and suggest that RHAMM is significantly associated

with ERK expression and may function collaboratively with

MAP kinase cascade in breast cancer progression. The role of

RHAMM in regulating ERK function is currently being inves-

ligated in our laboratories.

ras mutations appear to be early events in human coborectal

tumor development (7), but results here suggest that overexpres-

sion of its downstream effectors, ERK and RHAMM, may be an

event in human breast cancer progression that occurs later and

therefore contributes to metastasis. This may be related to the

ability of ERK to control proteinase expression and cell motility

(28, 29, 34, 35). RHAMM overexpression has previously been

observed in human B cell malignancies (36-38), lung (1 1), and

pancreatic (10) carcinomas. In pancreatic cancer cell lines,

which are associated with mutations of ras, RHAMM mRNA

levels were elevated in cell lines showing a poorly differentiated

phenotype and high metastatic potential (10). RHAMM has also

previously been shown to contribute to cell motility, invasion,

and proliferation in fibroblasts in vitro (9, 13, 33, 39-41).

Recently, we have demonstrated that RHAMM is crucial to

controlling ras signaling (9) and, in particular, to regulation of

ERK expression. This likely contributes to the ability of

RHAMM to transform (9). Our study is the first to relate




T able 7 Correlation bet ween RHAMM and ra s expression in 250 human breast carcinomas

RHAMM

general

score

ras score”

0 1 2 3 Total

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4

14

8

7

7

0

0

0

6

12

11

21

10

1

1

0

1

14

13

13

13

4

3

0

5

10

15

16

28

7

3

2

16

50

47

57

58

12

7

2

Total

Average

40

0.99

62

1.19

61

1.39

86

1.55

249

1.33

C’ r 0.27 (P = 0.038).

Table 8 RHAMM expression c orrelated with ERK expression

RHAMM VS. ERK r P

RHAMM (+E9) vs. ERK 0.26 0.0088RHAMM (-E9) vs. ERK 0.43 <0.0001

RHAMM total vs. ERK 0.38 <0.0001

Table 9 Functional relation ship betwee n RHAMM and ERK

Expression Case no. Median grade P”

RHAMM +++, ERK +++ 21 7

RHAMM + +, ERK + + + 30 6 0.034

C’ Mann-Whitney U test.

RHAMM to breast cancer and to assess its prognostic value as

a human tumor oncogene.

RHAMM occurs on the cell surface, in the cytoplasm, and

in the nucleus (11, 13, 14, 16, 38). We have previously proposed

that RHAMM, which is not an integral membrane protein, may

associate with the cell surface peripherally (as does another

extracellular matrix receptor, the laminin/elastin receptor corn-

plex; Ref. 42) or by a glycosylphosphatidylinositol modification

(43). RHAMM regulates src activation (41) and focal adhesion

kinase phosphorylation status (33), events that have an impact

upon the organization of the cytoskeleton and may also contrib-

ute to the activation of ERK through ras/focal adhesion kinase

interaction (41, 44). The relationship between cell surface asso-

ciated RHAMM and cytoplasmic and nuclear RHAMM with

ERK activation is under investigation in our laboratory.

A role of HA and its receptors in tumor progression and ras

signaling has previously been noted (45-47). In addition, cor-

relative studies have linked expression of receptors and HA to

human tumors (48-52). A direct link between HA and the HA

receptors CD44 and RHAMM in experimental tumor cell

growth has been demonstrated by studies showing that muta-

tions of the HA binding sites within these proteins ablates their

ability to promote cell growth (9, 46, 53). Furthermore, there is

a strong link between CD44 variant expression and human

tumor progression, although it is not yet clear whether this

relationship involves its HA binding capability (46, 47, 54-57).

In the case of breast cancer, Matsumura and Tarin (58) first

reported overexpression of CD44v gene products in human

breast cancer tissues and suggested that the expression of

CD44v may be a useful marker for the assessment of the

metastatic potential of the tumor. However, divergent conclu-

sions have been made in a number of later studies that used both

RT-PCR and immunohistochemical techniques. Although some

studies (59-61) support the results reported by Matsumura and

Tarin (58), other studies could not establish correlations be-

tween the expression of CD44v6 and the metastasizing capabil-

ity of breast cancer (62), between the expression of CD44v6 and

patient survival (63, 64), or between the expression of CD44v6

and poor prognosis parameters (65). The precise role that CD44

and RHAMM play in regulating the ras signaling cascade re-

quires further study.

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1998;4:567-576. Clin Cancer Res C Wang, A D Thor, D H Moore, 2nd, et al. in breast cancer progression.mitogen-activated protein kinase and is a significant parameterthat regulates ras signaling, correlates with overexpression of The overexpression of RHAMM, a hyaluronan-binding protein

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