Open Access

Increased RHAMM expression relates to ovarian cancer progression

Journal of Ovarian Research201710:66

https://doi.org/10.1186/s13048-017-0360-1

Received: 19 May 2017

Accepted: 7 September 2017

Published: 27 September 2017

Abstract

Background

Elevated hyaluronan-mediated motility receptor (RHAMM) has been reported to contribute to disease progression, aggressive phenotype and poor prognosis in multiple cancer types, however, RHAMM’s role in ovarian cancer (OC) has not been elucidated. Therefore, we sought to evaluate the role for RHAMM in epithelial OC.

Results

Despite little to no expression in normal ovarian surface epithelium, western immunoblotting, immunohistochemical staining and enzyme linked immunosorbent assay showed elevated RHAMM levels in clinical tissue sections, omental metastasis and urine specimens of serous OC patients, as well as in cell lysates. We also found that RHAMM levels increase with increasing grade and stage in serous OC tissues and that RHAMM localizes to the apical cell surface and inclusion cysts. Apical localization of RHAMM suggested protein secretion which was validated by detection of significantly elevated urinary RHAMM levels (p < 0.0001) in OC patients (116.66 pg/mL) compared with normal controls (8.16 pg/mL). Likewise, urinary RHAMM levels decreased following cytoreductive surgery in OC patients suggesting the source of urinary RHAMM from tumor tissue. Lastly, we validated RHAMM levels in OC cell lysate and found at least 12× greater levels compared to normal ovarian surface epithelial cells.

Conclusion

This pilot study shows, for the first time, that RHAMM may contribute to OC disease and could potentially be used as a prognostic marker.

Keywords

Hyaluronan-mediated motility receptorOvarian cancerImmunohistochemistry

Background

Commonly described as the “silent killer”, ovarian cancer (OC) is the fifth leading cause of cancer related deaths in women and it has the highest mortality of all gynecological malignancies [1]. Roughly one in 70 women will develop OC in her lifetime with only a 45% 5 year survival [2]. When disease has undergone metastatic spread, the survival rate drastically decreases from > 90% in early stage disease to less than 30% in late stage [1]. OC lethality is largely due to ambiguous symptoms, emergence of drug resistance, disease reoccurrence and lack of reliable screening methods all leading to late stage diagnosis. This underscores the need to improve our understanding of this disease and its etiology. Delineating key players driving disease would help elucidate potential molecular targets for treatment as well as for monitoring and detecting disease.

Receptor for hyaluronan-mediated motility (RHAMM) belongs to a group of hyaladherins, which share a common ability to bind to hyaluronan (HA). Based on subcellular localization, RHAMM performs multiple functions. Intracellularly, RHAMM is involved in microtubule spindle assembly, thereby contributing to cell cycle progression [3]. On the extracellular surface, RHAMM forms a trimeric complex with cluster differentiation 44 (CD44) and HA to activate cell signaling pathways that promote migration, invasion and cell proliferation [4]. While RHAMM is overexpressed in hematological malignancies and solid tumors arising from prostate [5], bladder [6] and breast [7], it is not known whether RHAMM contributes to OC. Although minimally expressed in normal tissue, elevated RHAMM in breast cancer (BC) and colorectal cancer (CRC) is associated with poor clinical outcome and more a aggressive cancer phenotype [7, 8]. Herein, we sought to determine if RHAMM similarly contributes to OC progression.

Methods

Clinical specimens

With University of South Florida Institutional Review Board approval (studies #Pro00003119 & 4739) and patient consent, tissues were collected from a cohort of women who had undergone primary surgery with complete surgical staging for epithelial ovarian cancer (EOC) or low malignant potential (LMP) tumors as defined by as benign but still containing abnormal cells, at the Moffitt Cancer Center and the University of South Florida (Table 1). This gynecologic oncology database was also used to select women who had undergone oophrectomy due to cystadenoma or had their ovaries removed for unrelated pathology. All tissue specimens were fixed with 10% formalin, paraffin-embedded, sectioned and stained with hematoxylin and eosin (H & E). The slides were reviewed by pathologists (AC, SVN) to confirm histologic diagnosis according to the International Federation of Gynecology and Obstetrics (FIGO) classification system.
Table 1

Summary of the patient cohort

 

N = 33 Patients

LGSC

HGSC

Normal

5

  

Serous OC

22

7

15

Stage

 I

2

2

0

 II

3

0

3

 III

9

3

6

 IV

8

2

6

Other

Normal Fallopian Tube

6

N/A

N/A

Breast Cancer

2

1

1

University of South Florida Institutional Review Board approval and patient consent was obtained for prospective (studies #Pro00003119, #Pro00000903) and retrospective (study #106004) collection of urine samples. Annonymized urine samples from healthy controls (N = 29), patients with benign gynecological pathology (N = 32) or OC (N = 150) were released for research from the Moffitt Cancer Center and the University of South Florida. All samples were centrifuged at 3000 x g and the supernatant was aliquoted and frozen at −20 °C before analyses were conducted.

Immunohistochemistry

For immunohistochemical (IHC) studies, formalin-fixed paraffin sections were cut at 3 μm and dried overnight at room temperature (RT) then deparaffinized and rehydrated. Sections were incubated in BLOXALL™ Blocking Solution (Vector Laboratories, Burlingame, Ca) for 20 min to block endogenous peroxidase activity. Antigen retrieval was achieved by placing slides in 1× solution Antigen Unmasking Solution (Vector Laboratories, Burlingame, Ca) brought to a boil and maintained at 95 °C for 30 min on a hot plate. Specimens were then immunostained using rabbit anti-human CD168 (Ca#:PA5–32309 ThermoFisher Scientific, Waltham, MA) primary antibody (1:100) for 1 h and Vectastain® Elite ABC Kit (Vector Laboratories), Dako’s EnVision™ and HRP Rabbit (DAB+) kit according to the manufacturer’s instructions, then counterstained with modified Mayer’s haematoxylin, dehydrated through graded alcohol, cleared with xylene, and mounted with resinous mounting medium. To control variability, all samples were stained at the same time and with the same lot of reagents. BC was used as an internal positive control while negative controls were obtained by substitution of primary antibody with normal rabbit serum.

Evaluation of RHAMM staining

A minimum of 100 cells were counted at a final magnification of 400X per tissue section. Immunostaining of RHAMM was scored based on average percent of positive epithelial cells (0, negative or no staining; 1 +, < 30%; 2 +, 30–50% and; 3 +, > 50%) and on staining intensity (negative, weak, moderate and strong). Cellular localization of RHAMM was also assessed as cytoplasmic, membranous, nuclear or stromal.

Images

Images were acquired on a digital Olympus DP-20 camera under a Leica dmire2 microscope. Olympus micro imaging software CellSens platform was used to acquire and process images. Images were taken with a final magnification of 100 × and 400 ×.

Western blot (WB)

For urinary analysis, normal and OC urine samples of equivalent volume were centrifuged at 16,000 x g using 30,000 kDa microfilters (Millipore, Bedford, MA) to concentrate the urine specimens. Concentrated urine samples were subjected to a Bradford protein assay and 30μg of protein were electrophoresed via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane. Membranes were blocked for 1 h at RT in 5% milk in tris-buffer saline with tween. Membranes were incubated overnight at 4 °C in monoclonal rabbit anti-CD168 RHAMM antibody (Ca# ab124729 abcam®, Cambridge, MA) and then incubated for 1 h at RT in goat anti-rabbit HRP conjugated antibody (Thermo Fisher Scientific, Waltham, MA). Protein bands were visualized using SuperSignal West Femto Substrate (Thermo Fisher Scientific, Waltham, MA) followed by densitometric analysis using Image Studio Lite Version 5.0 software program.

For analysis of RHAMM levels in cultured cells, the SV 40-Large T-Ag-transfected human OSE (HIOSE-118 and HIOSE-121) and OC (OVCAR5, OV90, and SKOV3) cells were cultured in Medium 199/MCDB 105 (Sigma, St. Louis, MO) with 5% fetal bovine serum and gentamicin. All cells were incubated at 37 °C with 5% CO2. Cells were washed in PBS, trypsinized, pelleted, and washed 1–2 times in cold PBS. Cells were lysed in CHAPS buffer and 30 μg of protein was separated via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene fluoride (PVDF) membranes and blocked in 5% milk in Tween 20-Tris buffered saline. Blots were incubated in their respective primary antibodies overnight, followed by incubation with a horseradish peroxidase-(HRP-) conjugated secondary antibody (Fisher, Pittsburgh, PA), and developed via enhanced chemiluminescence substrate (ECL) (Pierce/Fisher, Pittsburgh, PA) followed by densitometric analysis using Image Studio Lite Version 5.0 software program. Antibodies used were monoclonal rabbit anti-CD168 RHAMM antibody (Ca# ab124729 abcam®, Cambridge, MA) previously validated by Coulson-Thomas et al. doi:10.1074/jbc.M114.557447 [9].

Human HMMR/CD168/ RHAMM sandwich enzyme-linked immunosorbent assay (ELISA)

RHAMM ELISA (LifeSpan BioSciences, Inc., Seattle, WA) was performed according to the manufacturer’s recommended instructions for urine sample specimens. Prior to performing the experiments, all samples were thawed to RT and centrifuged to remove particulate matter. Plates were read using a microplate reader (BioTek ELx800, Winooski, VT) with a 450 nm wavelength filter.

Statistical analysis

Samples for RHAMM ELISA were run in duplicate and concentrations calculated as per manufacturer’s protocol. Data were subjected to the student T-Test for determination of statistical differences. p ≤ 0.05 was considered statistically significant.

Results

RHAMM is overexpressed in OC

Immunological staining was performed on 41 tissue sections from 33 women. The sample population provided sections of normal ovarian surface epithelium (OSE) (n = 5), serous OC (n = 22), omental metastasis (n = 7), lymph node metastasis (n = 1), and normal fallopian tube (FT) (n = 6). Though these samples comprise a small pilot study, they are representative of a typical clinical practice with regards to histological distribution (Table 1). BC was used as a positive control (n = 2).

Overall, we found that 91% (20/22) of serous OC stained positively for RHAMM with levels of staining intensity ranging from weak (< 30% from each field, N = 4), moderate (30–50%, N = 7) to strong (> 50%, N = 9) while 0% (0/5) of normal OSE stained for RHAMM. (Fig. 1, Table 2) Staining patterns, as depicted by intense, punctate/diffuse cytoplasmic staining, seen in BC positive control were consistent with previous reports of RHAMM in BC where intense staining is predominately in the cytoplasm and nucleus, but negative in the stroma [10, 11].
Fig. 1

RHAMM staining is elevated in OC tissue specimens. Representative photographs of IHC staining for RHAMM [anti-CD168 polyclonal antibody (PA5–32309) with a 1:100 dilution] in (b). normal, (c). LMP (d, e, f). LGSC and (g, h). HGSC. High grade BC was used as a positive control (a). Control sections were incubated with non-immune serum. Asterisks indicate stroma*. Original magnification 100× and 400×

Table 2

Quantification of RHAMM IHC

   

% Staining

Staining intensity

 

N

Positive

< 30%

30–50%

> 50%

Weak

Moderate

Strong

Normal

5

0

      

Serous OC

22

20/22

4/22

7/22

9/22

5/22

6/22

9/22

Grade

 Low

7

6/7

2/7

3/7

1/7

3/7

2/7

1/7

 High

15

14/15

2/15

4/15

8/15

2/15

4/15

8/15

Stage

 I

2

2/2

1/2

1/2

0

1/2

1/2

0

 II

3

3/3

1/3

1/3

1/3

0/3

2/3

1/3

 III

9

8/9

1/9

3/9

4/9

3/9

1/9

4/9

 IV

8

7/8

1/8

2/8

4/8

1/8

2/8

4/8

Fallopian Tube

6

6/6

0

1/6

5/6

1/6

2/6

3/6

RHAMM expression was predominately localized in the cytoplasm in 91% (20/22) of the serous OC specimens while the surrounding stromal tissue remained negative (Fig. 1, Table 3). Further, membranous staining was seen in 27% (6/22) of serous OC (Fig. 1d, e, f &h) and 18% (4/22) of serous OC specimens demonstrated nuclear staining (Fig. 1h). Low-grade serous carcinoma (LGSC) displayed membranous RHAMM staining localized to the apical cell surface (Fig. 1d, e, f & h). Interestingly, we noted RHAMM staining in OC cysts (Fig. 1d&f).
Table 3

Subcellular localization of RHAMM

Localization

N

Positive

Cytoplasm

Membrane

Nuclear

Stroma

Normal

5

0

    

Serous OC

22

20/22

20/22

6/22

4/22

1/22

Grade

 Low

7

6/7

6/7

1/7

1/7

1/7

 High

15

14/15

14/15

5/15

3/15

0

Stage

 I

2

2/2

2/2

0

0

0

 II

3

3/3

3/3

1/3

1/3

0

 III

9

8/9

8/9

3/9

2/9

0

 IV

8

7/8

7/8

2/8

1/8

1/8

Fallopian Tube

6

6/6

5/6

5/6

2/6

2/6

RHAMM intensity in primary serous OC tumors increases with grade and stage

High-grade serous carcinoma (HGSC) sections displayed intense punctate staining, LGSC displayed mostly weak, diffuse staining while LMP sections had almost no staining (Fig. 1, Table 2). Although percentages of positive staining was similar in LGSC (6/7 or 86%) and HGSC (14/15 or 93%) serous OC, increased staining intensity correlated with increasing grade. Strong RHAMM staining intensity was calculated as 14% and 53% of LGSC versus HGSC specimens respectively (Fig. 1, Table 2). Additionally, we found a tendency for RHAMM staining intensity to increase with stage. Strong RHAMM staining intensity levels were shown to be 0%, 33%, 44% and 50% of total stage 1, 2, 3 and 4 specimens respectively (Table 2).

Incidentally, we also noted RHAMM staining in primary tumors and their respective metastases for eight serous OC patients. We found positive RHAMM staining in 88% (7/8) of the primary tumors and 88% (7/8) of their omental or 100% (1/1) of lymph node metastases. Similar RHAMM staining percentages, intensities and cellular localization were seen in primary tumors and their respective metastases among all 8 patient samples (Fig. 2, Table 4).
Fig. 2

Primary and metastatic OC stain equally for RHAMM. Representative photographs of IHC staining for RHAMM in multiple sections from within the same patient of (a) normal, (b) primary serous adenocarcinoma and (c) secondary omental metastatic tissue. Control sections were incubated with non-immune serum. Asterisks indicate stroma*. Original magnification 100× and 400×

Table 4

RHAMM immunostaining patterns shown in primary and metastatic tumors

Patient

Description

Grade

Staining

% Staining

Intensity

P1

Normal Ovary

0

Negative

P1

Primary OC

low

+

1

Weak

P1

Omental metastasis

low

+

1

Weak

P2

Primary OC

low

+

2

Weak

P2

Omental metastasis

low

+

2

Weak

P3

Normal Ovary

0

Negative

P3

Primary OC

high

+

3

Strong

P3

Omental metastasis

high

+

3

Moderate

P4

Primary OC

high

+

1

Weak

P4

Omental metastasis

high

+

1

Weak

P5

Primary OC

high

0

Negative

P5

Omental metastasis

high

0

Negative

P6

Primary OC

high

+

3

Strong

P6

Omental metastasis

high

+

3

Strong

P7

Primary OC

high

+

2

Moderate

P7

Omental metastasis

high

+

2

Moderate

P8

Primary OC

high

+

2

Moderate

P8

Lymph node metastasis

high

+

1

Weak

Normal fallopian tube epithelium (FTE) stains intensely for RHAMM

Compared to normal OSE, which failed to stain for RHAMM, we found 100% (6/6) positive RHAMM staining in all normal FTE specimens examined (Fig. 3). There was moderate to strong staining limited to the surface of the fimbrial epithelium, cytoplasm and nucleus while stromal elements (Fig. 3 asterisks) did not stain for RHAMM. Specifically, there was strong staining in the nucleus and apical surface of secretory cells (Fig. 3, solid arrows) while ciliated epithelial cells (Fig. 3, dotted arrow) showed moderate apical and cytoplasmic staining but negative nuclear staining (Fig. 3).
Fig. 3

Normal FTE stains intensely for RHAMM. Representative photographs of IHC staining for RHAMM in normal (a). OSE and (b, c). FT. Control sections were incubated with non-immune serum. Asterisks indicate stroma* and arrows indicate secretory (solid) and ciliated (dotted) FTE cells. Original magnification 100× and 400×

Urinary RHAMM levels are elevated in OC patients

Since RHAMM expression appeared localized to cytoplasm, cell surface membrane and especially at the apical cell surface in HGSC, normal FT and potentially within ovarian cystic fluids (Fig. 1), we sought to determine whether RHAMM could be secreted by OC cells and, thereby, be detected in bodily fluids. Urinary analysis of RHAMM protein levels from OC patients measured by WB indicated the presence of RHAMM protein in 6/9 (66.7%) urine samples from patients with serous OC, while RHAMM was undetectable in all (10/10) urine samples from healthy controls (Fig. 4a). For more quantitative analyses, ELISA studies revealed urinary RHAMM levels from serous OC (N = 150) averaged almost 15X higher than normal controls (N = 29) (Fig. 4b, Table 5). Urinary levels of RHAMM in normal controls averaged 8.16 pg/mL, compared to 116.66 pg/mL in OC patient urine (p < 0.0001). Additionally, ELISA measurements of urinary RHAMM from 32 women with benign gynecological diseases including ovarian cysts, uterine fibroids and teratomas averaged 12.47 pg/mL, slightly higher than normal controls, but still significantly lower than OC RHAMM levels (p < 0.0001) (Fig. 4b). Lastly, average urinary RHAMM levels were not significantly different between disease stage (Fig. 4c) or grade (Fig. 4d).
Fig. 4

Urinary RHAMM levels are elevated in OC patients. a Concentrated urine samples of equivalent volumes from normal controls (N = 10) and patients with serous OC (N = 9) were screened for RHAMM by WB. Membranes were incubated with anti- RHAMM (1:1000) overnight and visualized with enhanced chemiluminescent. BC cell lysate (MCF-7) was used for positive control. b Urinary samples were examined by ELISA for RHAMM levels in normal (N = 29), OC (N = 150) and benign gynecological disease (N = 32), according to disease stage (c) and (d) grade. Results are expressed as a mean pg/mL RHAMM ± SE and represented as a histogram where p ≤ 0.05 was considered statistically significant

Table 5

Clinical parameters of urinary patient cohort

Sample

N

Average urinary RHAMM (pg/mL)

Range lower limit

Range upper limit

p-value

Normal

29

8.16433368

−8.50288

54.43993895

 

Benign

32

12.4764694

−1.029525763

85.95183057

p < 0.0001

OC

150

116.662456

−27.1929

1702.52388

p < 0.0001

STAGE

    

P = 0.249

 1

11

157.635708

14.12994

424.42521

 

 2

4

105.348346

37.508515

143.22621

 

 3

45

162.35244

−10.77318

1702.52388

 

 4

6

85.8344317

12.838485

175.459295

 

 5

11

87.5899564

22.47945

239.703255

 

GRADE

    

P = 0.280

 LGSC

23

142.265278

14.12994

424.42521

 

 HGSC

27

141.993346

−10.77318

1702.52388

 

*Values are expressed as an average urinary RHAMM level (pg/mL) where p < 0.05 was considered statistically significant

Urinary RHAMM levels decrease after cytoreductive surgery in OC

Urinary RHAMM levels were also measured by ELISA in 10 OC patients (Fig. 5a) and two patients with LMP ovarian tumors (Fig. 5b) immediately prior to initial cytoreductive surgery, within 2 weeks of cytoreductive surgery and, where possible, at a t3 month post-operative follow-up. We found up to 89% reduction in urinary RHAMM levels post-cytoreductive surgery compared to pre-cytoreductive surgery in 70% (7/10) of OC patients 2 weeks post-surgery and in 80% (8/10) of OC patients 3 months post-surgery. An increase in urinary RHAMM levels 2 weeks post-cytoreductive surgery was noted in 30% (3/10) OC patients compared to pre- cytoreductive surgery urinary RHAMM levels. In contrast, urinary RHAMM measurements in patients with benign disease pre- and post-tumor reduction remained low (average 36 pg/mL), thereby remaining relatively unchanged with surgical intervention.
Fig. 5

Urinary RHAMM levels decrease after tumor debulking. Urinary levels of RHAMM were measured by ELISA from (a) 10 OC patients and (b) 2 patients with benign LMP ovarian tumors prior to tumor removal (black bars), within 2 weeks of debulking surgery (gray bars) and 3 months post debulking surgery (white bars) where available. Results are expressed as a mean pg/mL RHAMM ± SE and represented as a histogram where p ≤ 0.05 was considered statistically significant

RHAMM levels are elevated in OC cell lines

Validation of RHAMM expression in OC was performed measuring protein levels by WB of cellular RHAMM in OC and HIOSE cell cultures. RHAMM levels were consistently elevated as a single band at 85 kDa in OC cell lines (OV90 and OVCAR5) compared to IOSE cells (HIOSE-118 and HIOSE-121) and with BC cell line (MCF7) as a positive control (Fig. 6). Densitometric values confirm about 40× and 12× more RHAMM in OV90 cells compared to normal OSE and about 70× and 20× more RHAMM in OVCAR5 cells compared to control measured by Image Studio Lite computer software program.
Fig. 6

RHAMM is overexpressed in cultured OC cells. Protein levels of RHAMM were measured by WB in HIOSE-118, HIOSE-121, OV90 and OVCAR5 cells. WB was performed on protein cell lysates using monoclonal rabbit anti-CD168 RHAMM antibody at a 1:1000 dilution. Densitometric values were quantified using image analysis (Image Studio Lite Version 5.0) of target protein bands to β-actin levels where MCF-7 cells were used as a positive control

Discussion

Overall survival of OC has not improved for several years due to poor understanding of its pathogenesis, late diagnosis, emergence of drug resistance and lack of reliable biomarkers. Consequently, in order to better elucidate the etiology of this disease, the aim of this pilot study was to determine if, like other cancer types, RHAMM is overexpressed in OC and whether RHAMM could, likewise, promote OC progression.

We show for the first time that RHAMM expression is elevated in serous OC compared to normal OSE. We observed 91% of serous OC patients demonstrated positive RHAMM staining which was localized primarily to the cytoplasm, cell membrane, cystic fluid and, occasionally, the nucleus. RHAMM staining appeared to be specific to epithelial tumor cells since the stroma failed to stain. We also showed that RHAMM staining intensity increased with increasing cancer grade and showed a tendency to increase with stage. Levels of RHAMM appear to be dependent upon the extent of disease progression where LMP showed negligible to no staining, LGSC and early stage specimens demonstrated weak punctate staining while HGSC and late stage specimens typically demonstrated intense, but rather diffuse RHAMM staining. High levels of RHAMM in aggressive colorectal cancer tumor budding cells are associated with higher grade, poor survival, increased lymphatic invasion and nodal metastasis [12]. Additionally, invasive BC cell lines express higher levels of RHAMM [13] and IHC staining in 189 mammary carcinomas revealed that elevated RHAMM in lobular carcinomas is correlated with more invasive behavior and reduced overall patient survival time [11]. Therefore, increasing levels of RHAMM seen in OC could contribute to an invasive and progressive phenotype.

Since recent studies suggest that HGSC arises from the FTE [14], we also subjected normal FTE to IHC staining for RHAMM. Distinct molecular markers of HGSC include dysregulation of wild type p53 (wtp53), which is seen in about 96% of HGSC cases. Dysregulation of wtp53 can occur as gain of function (GOF) mutations, termed TP53 mutations. These mutations often lead to the acquisition of oncogenic functions, abrogating the cell cycle constraints controlled by wtp53 [15]. Since wtp53 is a negative transcriptional regulator of RHAMM protein [16] and HGSC datasets such as the CPTAC Data Portal, the Human Protein Atlas and GDC Data Portal revealed few RHAMM mutations in OC specimens, we speculate that dysregulation of wtp53 in OC could drive overexpression of RHAMM. Interestingly, RHAMM was strongly evident in all of the normal FT specimens examined, showing the most intense staining in the distal region of the FT and in nuclei of FTE secretory cells. Similar staining patterns of HGSC and secretory FTE cells align with the current theory of HGSC arising from tubal origins in contrast to LGSC, which is traditionally believed to arise in the OSE.

RHAMM is typically only loosely tethered to the cell membrane [3] suggesting it may be secreted by cells. We showed RHAMM localization to the apical OC cell surface and within cysts of serous OC further suggesting that RHAMM could be secreted. Therefore, we sought to determine whether RHAMM could be detected in bodily fluids. Specifically, we sought to determine whether elevated RHAMM levels could be detected in the urine of OC patients. Urinary markers are ideal in clinical settings since urine collection is simple, safe, non-invasive and cost effective. In addition, urinary proteins retain high stability and urinary filtration precludes the presence of large proteins found in serum, such as albumin, which can confound test results.

In this pilot study, we consistently found elevated urinary RHAMM levels in OC patients that were significantly higher than normal healthy controls and women with benign gynecological disease (*P < 0.0001). While most patients with benign gynecological disease did not demonstrate elevated urinary RHAMM, elevated urinary RHAMM was observed in a patient with uterine fibroids and a patient with endometrioma. During inflammation, HA levels increase within the microenvironment which, in turn, promotes increased RHAMM secretion [17] and involvement of RHAMM in the inflammatory process [18]. Consequently, inflammatory benign gynecologic conditions, as may have been present in these two patients, could result in a transient increase in urinary RHAMM levels.

While urinary RHAMM levels were essentially unchanged in patients with benign disease following cytoreductive surgery, reduced urinary RHAMM levels after cytoreductive surgery in OC patients suggests that RHAMM is produced and secreted by OC cells. However, it is important to note that 35.8% of patients experience post-surgical infections following cytoreductive surgery [19] and this may account for increased urinary RHAMM levels in the patients who experienced elevated RHAMM levels at their 2 week follow-up. Nonetheless, utilizing RHAMM for monitoring disease after surgical debulking as a prognostic marker could provide a useful clinical tool for monitoring disease reoccurrence.

RHAMMs’ α-helical and coiled structure confers a hydrophilic and water soluble protein profile [20] providing a mechanism by which RHAMM might be present in bodily fluids. Given that glomerular filtration typically excludes high molecular weight proteins from the urine, we were surprised to detect full length 85 kDa RHAMM protein in OC patient urine by WB. However, our results are in keeping with others who have also reported high molecular weight (MW) proteins in urine of women at high risk for BC including matrix metallopeptidase-2, matrix metallopeptidase-9 (MMP9) and MMP9/neutrophil gelatinase-associated lipocalin complex (MW: 72 kDa, 92 kDa and 115 kDa respectively) [21]. High MW proteins present in the urine are commonly associated with renal dysfunction [22]. Donadio et al. (2003) reported renal impairment in both early and late stage OC showing at least a 10% impairment of glomerular filtration rate and creatinine clearance in 30% of stage 1, 50% of stage 2, 56% of stage 3 and 64% of stage 4 OC patients [22] suggesting that renal impairment due to disease could enable urinary transport of full-length RHAMM. Alternately, despite lacking a secretory signal peptide sequence, RHAMM is commonly secreted from cells by an as yet unknown mechanism [23]. Consequently, RHAMM may bypass glomerular filtration allowing its transport into the urine by a glomerular-independent mechanism.

When analyzing clinical parameters, average urinary RHAMM levels were independent of stage and grade. This is in contrast to our IHC studies where we found an increase in RHAMM by grade and a slight trend for RHAMM to increase by stage. Assmann et al. (2001) showed significantly higher expression of an intracellular splice variant of RHAMM than its cell surface splice variant in BC cells and proposed differential RHAMM splice variant expression for BC pathogenesis [24]. Similarly, the transition from LGSC to HGSC OC may be accompanied by preferential RHAMM splice variant expression so that increased RHAMM staining intensity and cytoplasmic localization noted with increasing grade and stage may reflect a shift away from membranous/extracellular RHAMM production towards increased production of intracellular RHAMM.

Lastly, WB confirmed significantly elevated RHAMM protein in OC cell lysates compared to normal HIOSE cell lines. Therefore, OC cell culture may provide a model system to delineate the molecular and mechanistic function by which RHAMM contributes to OC.

Conclusion

This pilot study is the first to show RHAMM expression in OC as well as a potential prognostic clinical impact for urinary RHAMM levels. Further, the apparent relationship between increased RHAMM production with increasing tumor grade and stage suggests that RHAMM contributes to OC progression. Lastly, given the shared characteristics of HGSC and FTE reported to date, further studies are clearly warranted to discern the degree to which RHAMM may also be a member of this shared molecular profile.

Abbreviations

BC: 

Breast cancer

CD44: 

Cluster differentiation 44

CRC: 

Colorectal cancer

ELISA: 

Enzyme-linked immunosorbent assay

EOC: 

Epithelial ovarian cancer

FT: 

Fallopian tube

FTE: 

Fallopian tube epithelium

GOF: 

Gain of function

H&E: 

Hematoxylin and eosin

HA: 

Hyaluronan

HGSC: 

High-grade serous cancer

IHC: 

Immunohistochemical

LGSC: 

Low-grade serous cancer

LMP: 

Low malignant potential

MMP9: 

Matrix metallopeptidase-9

MW: 

Molecular weight

OC: 

Ovarian cancer

OSE: 

Ovarian surface epithelium

RHAMM: 

Receptor for hyaluronon-mediated motility

RT: 

Room temperature

TP53: 

Mutated p53

WB: 

Western immunoblot

WTP53: 

Wild type p53

Declarations

Acknowledgements

This work was supported, in part, by an Ovarian Cancer Research Fund GRT 10935 (SVN, PAK), US Department of Defense Idea Development Award OC060142 (PAK) and a Fifth Third Foundation Cancer Biology Fellowship (STB).

Funding

This work was supported, in part, by an Ovarian Cancer Research Fund GRT 10935 (SVN, PAK) and a Fifth Third Foundation Cancer Biology Fellowship (STB).

Availability of data and materials

The data supporting the conclusions of this article are included within this manuscript.

Authors’ contributions

The contributions of each author have been significant: STB performed the experiments, analyzed the data, wrote the manuscript and prepared figures for this manuscript. MSH, AC and SVN procured and reviewed de-identified clinical specimens while AK performed the clinical statistical analyses. As PI, PAK developed and oversaw this project from its planning through execution and preparation of the present manuscript. All authors read and approved the final manuscript.

Ethical approval and consent to participate

This study was reviewed and approved by the University of South Florida Institutional Review, Division of Research Compliance for both prospective and retrospective sample collection. All patients gave informed consent for prospective participation in this study.

Consent for publication

Not applicable.

Competing interests

STB and PAK are co-authors on a provisional US patent entitled “Detection of Ovarian Cancer by Elevated Urinary Levels of Rhamm” USF Ref. No. 16A034PR. The remaining authors have no conflicts of interest to report.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Pathology & Cell Biology, University of South Florida, Morsani College of Medicine
(2)
Gynecologic Oncology, Moffitt Cancer Center
(3)
Department of Internal Medicine, University of South Florida
(4)
Department of Obstetrics & Gynecology, University of South Florida

References

  1. Institute NC. Surveillance, epidemiology and end results program. http://seer.cancer.gov/. Accessed 26 June 2016.
  2. Cannistra SA. Cancer of the ovary. N Engl J Med. 2004;351(24):2519–29. doi:10.1056/NEJMra041842.View ArticlePubMedGoogle Scholar
  3. Entwistle J, Hall CL, Turley EA. HA receptors: regulators of signalling to the cytoskeleton. J Cell Biochem. 1996;61(4):569–77. doi:10.1002/(SICI)1097-4644(19960616)61:4&lt;569::AID-JCB10&gt;3.0.CO;2-B. View ArticlePubMedGoogle Scholar
  4. Zhang S, Chang MC, Zylka D, Turley S, Harrison R, Turley EA. The hyaluronan receptor RHAMM regulates extracellular-regulated kinase. J Biol Chem. 1998;273(18):11342–8.View ArticlePubMedGoogle Scholar
  5. Gust KM, Hofer MD, Perner SR, et al. RHAMM (CD168) is overexpressed at the protein level and may constitute an immunogenic antigen in advanced prostate cancer disease. Neoplasia. 2009;11(9):956–63. doi:10.1593/neo.09694.View ArticlePubMedPubMed CentralGoogle Scholar
  6. Niedworok C, Kretschmer I, Röck K, et al. The impact of the receptor of hyaluronan-mediated motility (RHAMM) on human urothelial transitional cell cancer of the bladder. PLoS One. 2013;8(9):e75681. doi:10.1371/journal.pone.0075681.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Veiseh M, Kwon DH, Borowsky AD, et al. Cellular heterogeneity profiling by hyaluronan probes reveals an invasive but slow-growing breast tumor subset. Proc Natl Acad Sci U S A. 2014;111:E1731–9. doi:10.1073/pnas.1402383111.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Koelzer VH, Huber B, Mele V, et al. Expression of the hyaluronan-mediated motility receptor RHAMM in tumor budding cells identifies aggressive colorectal cancers. Hum Pathol. 2015;46(11):1573–81. doi:10.1016/j.humpath.2015.07.010.
  9. Coulson-Thomas VJ, Gesteira TF, Hascall V, Kao W. Umbilical cord mesenchymal stem cells suppress host rejection: the role of the glycocalyx. J Biol Chem. 2014;289(34):23465–81. doi:10.1074/jbc.M114.557447.View ArticlePubMedPubMed CentralGoogle Scholar
  10. Wang C, Thor AD, Moore DH, et al. 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. Clin Cancer Res. 1998;4(3):567 LP-576. http://clincancerres.aacrjournals.org/content/4/3/567.abstract.Google Scholar
  11. Assmann V, Gillett CE, Poulsom R, Ryder K, Hart IR, Hanby AM. The pattern of expression of the microtubule-binding protein RHAMM/IHABP in mammary carcinoma suggests a role in the invasive behaviour of tumour cells. J Pathol. 2001;195(2):191–6. doi:10.1002/path.941.View ArticlePubMedGoogle Scholar
  12. Koelzer VH, Bettina H, Valentina M, et al. Expression of the hyaluronan acid mediated motility receptor RHAMM in tumor budding cells identifies aggressive colorectal cancers. Hum Pathol Accept Publ 2015. doi:10.1016/j.humpath.2015.07.010.
  13. Hamilton SR, Fard SF, Paiwand FF, et al. The hyaluronan receptors CD44 and Rhamm (CD168) form complexes with ERK1,2 that sustain high basal motility in breast cancer cells. J Biol Chem. 2007;282(22):16667–80. doi:10.1074/jbc.M702078200.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Przybycin CG, Kurman RJ, Ronnett BM, Shih LM, Vang R. Are all pelvic (nonuterine) serous carcinomas of tubal origin? Am J Surg Pathol. 2010;34(10):1407–16. doi:10.1097/PAS.0b013e3181ef7b16.View ArticlePubMedGoogle Scholar
  15. Brachova P, Thiel KW, Leslie KK. The consequence of oncomorphic TP53 mutations in ovarian cancer. Int J Mol Sci. 2013;14(9):19257–75. doi:10.3390/ijms140919257.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Sohr S, Engeland K. RHAMM is differentially expressed in the cell cycle and downregulated by the tumor suppressor p53. Cell Cycle. 2008;7(21):3448–60. doi:10.4161/cc.7.21.7014.View ArticlePubMedGoogle Scholar
  17. Turley EA, Noble PW, Bourguignon LYW. Signaling properties of hyaluronan receptors. J Biol Chem. 2002;277(7):4589–92. doi:10.1074/jbc.R100038200.View ArticlePubMedGoogle Scholar
  18. Nikitovic D, Tzardi M, Berdiaki A, Tsatsakis A, Tzanakakis GN. Cancer microenvironment and inflammation: role of hyaluronan. Front Immunol. 2015;6:169. doi:10.3389/fimmu.2015.00169.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Valle M, Federici O, Carboni F, et al. Postoperative infections after cytoreductive surgery and HIPEC for peritoneal carcinomatosis: proposal and results from a prospective protocol study of prevention, surveillance and treatment. Eur J Surg Oncol. 2014;40(8):950–6. doi:10.1016/j.ejso.2013.10.015.View ArticlePubMedGoogle Scholar
  20. Tolg C, McCarthy JB, Yazdani A, Turley EA. Hyaluronan and RHAMM in wound repair and the “Cancerization” of Stromal tissues. Biomed Res Int. 2014;2014:103923. doi:10.1155/2014/103923.View ArticlePubMedPubMed CentralGoogle Scholar
  21. Pories SE, Zurakowski D, Roy R, et al. Urinary metalloproteinases: noninvasive biomarkers for breast cancer risk assessment. Cancer Epidemiol Biomark Prev. 2008;17(5):1034–42. doi:10.1158/1055-9965.EPI-07-0365.View ArticleGoogle Scholar
  22. Donadio C, Lucchesi A, Ardini M, Cosio S, Gadducci A. Renal impairment in patients with ovarian cancer. Eur J Obstet Gynecol Reprod Biol. 2003;106(2):198-202. doi:10.1016/S0301-2115(02)00234-8.
  23. Maxwell CA, McCarthy J, Turley E. Cell-surface and mitotic-spindle RHAMM: moonlighting or dual oncogenic functions? J Cell Sci. 2008;121:925–32. doi:10.1242/jcs.022038.View ArticlePubMedGoogle Scholar
  24. Assmann V, Marshall JF, Fieber C, Hofmann M, Hart IR. The human hyaluronan receptor RHAMM is expressed as an intracellular protein in breast cancer cells. J Cell Sci. 1998;1694: 1685-1694.Google Scholar

Copyright

© The Author(s). 2017

Advertisement