TOTAL NUMBER OF PAGES: NUMBER OF FIGURES · 12/07/2011 · Author manuscripts have been peer...

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Nerve growth factor links oral cancer progression, pain, and cachexia

Yi Ye1, Dongmin Dang2, Jianan Zhang1, Chi Tonglien Viet3,

David K. Lam2, John Dolan1,4, Jennifer Gibbs5, and Brian L. Schmidt1,3

1Bluestone Center for Clinical Research, New York University

2Department of Oral and Maxillofacial Surgery, University of California San Francisco

3Department of Oral Maxillofacial Surgery, New York University

4Department of Orthodontics, New York University

5 Department of Endodontics, New York University

Running title: Anti-NGF as a therapy in head and neck cancer

Keywords: NGF, allodynia, cancer pain, weight loss, tumor growth

TOTAL NUMBER OF PAGES: 29

NUMBER OF FIGURES: 6

Word count: 5853

*Corresponding author:

Brian L. Schmidt DDS, MD, PhD

Bluestone Center for Clinical Research, New York University College of Dentistry

421 First Avenue, 233W, New York, New York 10010

Email: [email protected]

Tel: 212-998-9543 Fax: 212-995-4843

The authors have no conflicts of interest to declare.

This work was funded by NIH/NIDCR R21 DE018561

on January 25, 2021. © 2011 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 12, 2011; DOI: 10.1158/1535-7163.MCT-11-0123

http://mct.aacrjournals.org/

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Abstract

Cancers often cause excruciating pain and rapid weight loss, severely reducing quality

of life in cancer patients. Cancer-induced pain and cachexia are often studied and

treated independently, although both symptoms are strongly linked with chronic

inflammation and sustained production of pro-inflammatory cytokines. Since nerve

growth factor (NGF) plays a cardinal role in inflammation, and pain, and because it

interacts with multiple pro-inflammatory cytokines, we hypothesized that NGF acts as a

key endogenous molecule involved in the orchestration of cancer-related inflammation.

NGF might be a molecule common to the mechanisms responsible for clinically

distinctive cancer symptoms such as pain and cachexia as well as cancer progression.

Here we reported that NGF was highly elevated in human oral squamous cell carcinoma

tumors and cell cultures. Using two validated mouse cancer models, we further

demonstrated that NGF blockade decreased tumor proliferation, nociception, and

weight loss by orchestrating pro-inflammatory cytokines and leptin production. NGF

blockade also decreased expression levels of nociceptive receptors TRPV1, TRPA1,

and PAR-2. Together, these results identified NGF as a common link among

proliferation, pain, and cachexia in oral cancer. Anti-NGF could be an important

mechanism-based therapy for oral cancer and its related symptoms.




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Introduction

Pain and cachexia significantly impair function and degrade quality of life in

patients suffering from cancer (1-7). Pain control and weight maintenance are especially

challenging in patients with head and neck (oral) cancer. Many oral cancer patients

suffer from symptoms that are more severe than symptoms produced by other cancers

(4-7). Oral cancer patients often experience difficulty with eating, drinking, swallowing,

and speaking. Despite recent advances in treatment, pain control and weight

maintenance persist as two important clinical challenges. Cancer pain and cachexia are

usually assessed and treated as separate, unrelated entities (1). However, these

symptoms may share the same underlying mechanism since both are linked to chronic

inflammation and share common pro-inflammatory mediators (1, 8). Currently, such a

mechanism has not been elucidated for cancer pain and cachexia.

Nerve growth factor (NGF) is a key modulator of the neuro-endocrine immune

axis that serves diverse biological functions (9-13). In a variety of rodent cancer

models, NGF has been shown to play a direct role in tumor proliferation (13-15), and in

perineural invasion. Perineural invasion is a predictor of disease progression in oral

cancer (15). NGF has also been proposed as an important mediator of tumor-induced

bone pain secondary to prostate cancer (16-18). However, the effect of NGF on cancer

proliferation and pain is dependent on tumor type. For example, anti-NGF treatment has

been shown to reduce disease progression in breast cancer but has no effect in bone

cancer (14, 16, 17). Bone cancer pain, however, is attenuated by anti-NGF treatment in

mice (16-18). Oral cancer pain is usually caused by cancer originating from soft tissue

and clinically distinct from bone cancer pain. Oral cancer pain is exacerbated by




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function, generally does not arise spontaneously, and is not correlated with tumor size.

Even the smallest oral cancers can produce severe pain (4, 5). In contrast, bone cancer

pain is commonly spontaneous and incessant, and typically increases with disease

progression (16, 17). The effect of anti-NGF on oral cancer pain or proliferation is

unknown.

The role of NGF in the regulation of body weight also requires further study.

NGF has been shown to participate in glucose and lipid metabolism as well as feeding

behavior (19). Intraperitoneal injection of NGF stimulates the hypothalamic-pituitary-

adrenal axis and causes weight loss in rats (20). Patients with obesity have altered levels

of NGF (9, 19, 21). NGF might contribute to inflammation and metabolic disorders

associated with body weight changes (19, 21). Similarly, cancer cachexia is a complex

wasting syndrome comprised of inflammatory and metabolic disturbances (2). Pro-

inflammatory cytokines including tumor necrosis factor-alpha (TNF-�) and interleukin-

6 (IL-6) have been shown to induce cachexia by altering metabolism of lipids and

muscle proteins (2, 22, 23). NGF modulates the expression and release of these pro-

inflammatory cytokines (10, 24). Accordingly, NGF may play a role in cancer cachexia.

The varied pro-inflammatory and nociceptive effects of NGF suggest a key role

for NGF in cancer symptomatology. We hypothesize that NGF acts as a mediator of

proliferation, pain, and cachexia associated with oral cancer. In the present study, we

first quantified NGF release and expression in patients with oral squamous cell

carcinoma (SCC). We then employed two separate oral cancer models in mice to

demonstrate that anti-NGF reduces proliferation, pain, and weight loss. Interactions of




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NGF with pro-inflammatory cytokines and receptors involved in nociception were also

evaluated.

Materials and Methods

Cancer Patients

Immunohistochemistry for NGF in human tumors

Oral SCC on the affected side and normal epithelium from an anatomically-

matched area on the contra-lateral side of 14 oral cancer patients treated at the

University of California San Francisco (UCSF) Department of Oral & Maxillofacial

Surgery were obtained. Tissues were fixed with 4% paraformaldahyde (PFA),

dehydrated, embedded in paraffin, and cut into 8 μm sections. Microwave antigen

unmasking was done using Dako antigen retrieval solution (Dako, Carpinteria, CA).

Sections were then incubated with rabbit polyclonal antibodies against NGF (1:100;

Serotec, Inc., Raleigh, NC) for 2 hours. The specificity of NGF antibody was tested and

validated in our previous studies (15). Human submandibular gland was used as a

positive control as it is known to produce an abundance of NGF. Normal rabbit serum

containing mixed immunoglobulins at the concentration of the primary antibody was

used as a negative control on the salivary gland tissue specimens. Immunoreactions

were visualized with diaminobenzidine chromogen (Vector Laboratories, Peterborough,

UK) and counterstained with Mayer’s hematoxylin. This research protocol complies

with the Committee on Human Research at the University of California San Francisco.




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Reverse transcription-PCR to quantify NGF mRNA in human tumors

Oral SCC and anatomically-matched, contra-lateral normal oral epithelium from

11 oral cancer patients were surgically removed and immediately snap frozen in liquid

nitrogen and stored at -80 oC. Total RNA isolation of each sample was conducted with a

Qiagen DNA/RNA kit (Qiagen Inc., Valencia, CA) and 1μl samples were aliquoted for

RNA quantitative analysis. Reverse transcription was carried out using a High Capacity

cDNA Reverse Transcription Kit (Applied Biosystems Inc., Foster City, CA) on the

biometra thermocyler (template 10μl volumes per reaction). Quantitative real-time PCR

assays were performed in triplicate with a Taqman Gene Expression Assay kit (Applied

Biosystems Inc). The housekeeping gene �-gus was chosen as the internal control.

Controls consisted of total human brain RNA (~12ng/μl; Ambion, Austin, TX, USA)

and were negative in all runs. Relative quantification analysis of gene expression data

was conducted according to the 2-��CT method.

Cell culture of human oral SCC and control keratinocytes

Human oral cancer cells (HSC-3) were cultivated for inoculation into mouse models of

human cancer pain and proliferation. The HSC-3 squamous cell carcinoma cell line was

obtained from the Japanese Collection of Research Bioresources (JCRB). The JCRB

Cell Bank authenticated the cell line by using short tandem repeat analysis of loci with

the PCR-based PowerPlex1.2 system. Once we received the cells from JCRB the

cells were expanded and frozen stocks were prepared. For experiments, cells were used

at low passage for not more than 6 months before replenishing with fresh samples from




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the frozen stocks. Primary cultures of normal oral keratinocytes (NOK) harvested from

normal oral tissues, were cultured as previously described (25).

ELISA quantification of NGF in human oral cancer cells

HSC-3 cells and NOK were grown to confluence and then washed to remove

unattached cells. The media for both HSC-3 and NOK were replaced with DK-SFM and

incubated for an additional 72 hrs. The conditioned media was then removed,

centrifuged to remove cell debris, aliquoted and stored at -20 oC. Cells were lysed in

cold RIPA buffer containing protease inhibitor cocktails (Sigma-Aldrich, St. Louis,

MO). The lysate was centrifuged (13000 rpm for 5 min) and the supernatant was

removed, aliquoted, and stored at -20oC. NGF concentration was measured using the

NGF Emax Immunoassay ELISA kit (Promega, Madison, WI).

Proliferation assay with anti-NGF in human oral cells

To quantify the effect of NGF on cancer cell proliferation, 3×103 HSC-3 cells

were seeded in individual wells. Control groups received PBS (10μl) and experimental

groups received NGF neutralizing antibody (R&D systems, Minneapolis, MN) in PBS

(10μl of 0.025μg/ml). At predetermined time points (0, 24, 48, and 72h), 20�l of MTS

(Promega BioSciences, San Luis Obispo, CA) were added to each well for one hour and

incubated at 37oC. The samples were then quantified by MTS colorimetric assay at

490nm. The experiment was repeated in triplicate.




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Mouse Cancer Models

Behavioral mouse models of human oral cancer pain

Six to eight week-old female athymic, immunocompromised mice (BALB/c) were

purchased from Charles River Laboratories (Hollister, CA). They were housed in a

temperature-controlled room on a 12:12 light:dark cycle (0600–1800 h light), with ad

libitum access to food and water. The UCSF Committee on Animal Research approved

all procedures and researchers were trained under the Animal Welfare Assurance

Program.

Paw model: The paw-withdrawal cancer pain mouse model was produced as

previously described (26). Adult female nude mice were inoculated with 106 HSC-3

cells in 50 �l of DMEM and Matrigel™ into the plantar surface of the right hind-paw.

Tongue model: To create a mouse model that is more biologically homologous

with human oral cancer, mice were inoculated with 50 μl of 106 HSC-3 cells into the

floor of the mouth as previously described (27). The anatomic and functional features of

this mouse cancer model parallel those found in human patients with oral cancer (27).

Anti-NGF treatment and control groups

Paw. In the mouse paw-tumor model, anti-NGF antibody (Mab 256, R&D

Systems, San Jose, CA) (12.5 μg in 20 μl PBS) or vehicle control (20 μl PBS) was

injected into the right hind paw of mice starting on post-inoculation day (PID) 4

following the pain behavior measurement and twice a week thereafter until PID 21 (14).

Dosage of anti-NGF used was based on a study by Adriaenssens et al. (14). Mice were

randomly placed into four treatment groups: Group 1 received an injection of HSC-3




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cells and anti-NGF treatment (tumor + anti-NGF, n=7), Group 2 received an injection of

HSC-3 cells and PBS (vehicle control, tumor + PBS, n=7), Group 3 received an

injection of HSC-3 in the right paw and anti-NGF in the contra-lateral (CL) paw to see

whether anti-NGF has a systemic effect (tumor + CL-anti-NGF, n=5), Group 4 was

treated with anti-NGF to determine whether NGF is hypoanalgesic in naïve mice (naïve

+ anti-NGF, n=5). All groups of mice were briefly anesthetized with inhalational

isoflurane (Summit Medical Equipment Company, Bend, Oregon) during HSC-3

inoculation and drug treatments.

Tongue: In the mouse tongue-cancer model, two groups of mice were used. The

control group (n=10) received isotype IgG (50 μg in 50 μl PBS, R&D systems,

Minneapolis, MN). The anti-NGF treatment group (n=10) received 50 μg the anti-NGF

antibody in 50 μl PBS. All injections were intraperitoneal and administered twice per

week starting at post-inoculation day 13, when all mice exhibited visible tumor masses

and increased gnaw-time. We were concerned that repeated local injection of anti-NGF

into the tongue would affect the rodent’s eating and gnawing behavior so we chose a

systemic route of injection (intraperitoneal). Higher doses of systemic anti-NGF were

used in the tongue model compared to the dose given in the paw model to ensure

enough antibodies reached the tongue tumor.

Behavioral measurement

Paw-withdrawal assay: Testing was performed by an observer blinded to the

experimental groups as previously described (25). The paw withdrawal threshold was

measured using an electronic von Frey anesthesiometer (IITC Life Sciences, Woodland

Hills, CA). Paw withdrawal threshold was defined as the force in grams (mean of 8




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trials) sufficient to elicit a distinct paw withdrawal flinch upon application of a rigid

probe tip.

Dolognawmeter: The Dolognawmeter is a validated device/assay invented to

measure oral function and nociception in mice (27). Mice with tongue tumors were

evaluated twice per week with a dolognawmeter as previously described (27). In brief,

each mouse was placed into a confinement tube with two obstructing dowels in series.

The mouse voluntarily gnaws through the two dowels to escape from confinement

within the tube. Each obstructing dowel is connected to a digital timer. When the dowel

is severed by the gnawing of the mouse, the timer is automatically stopped and records

the duration of time to sever each of the two dowels. To acclimatize the mice and

improve consistency in gnawing duration, all mice were “trained” for 10 sessions in the

dolognawmeter. Training involves placing the animals in the device and allowing them

to gnaw through the obstructing dowels in exactly the same manner that they do so

during the subsequent experimental gnawing trials. A baseline gnaw-time value to sever

the second dowel was established for each mouse as the mean of the final three training

sessions. After baseline gnaw-times were established for each mouse, the mice were

inoculated with cancer cells.

Tumor size and body measurement

Mouse hind-paw volume was measured using a plethysmometer (IITC Life

Science, Woodland Hills, CA). Tongue tumor volume was calculated at the end of the

experiment by multiplying tumor length by width by thickness. Body weight was

recorded before each behavioral test. Mice did not show any significant weight changes

during baseline training trials.




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Tissue and blood processing

At the end of the experiment, mice with either cancer model were sacrificed

with isoflurane. Both tumor and normal paws were dissected and stored at -80oC in

preparation for NGF protein quantification. The paw samples were homogenized, lysed,

centrifuged, and the supernatant was removed. Total protein concentration in each

sample was determined using a BCA protein assay (Thermo Scientific, Rockford, IL).

NGF concentration was measured using the same method as previously described. In

mice with tongue cancer, blood was rapidly collected from the heart into EDTA coated

tubes. Plasma was separated with a centrifuge and stored at -20oC. Because adipose

tissue is the main source of leptin, abdominal fat was also rapidly dissected out and

stored at -80 oC. Following these manipulations, mice were perfused transcardially with

0.1 M PBS followed by 4% PFA. Trigeminal ganglia were harvested in preparation for

sensory receptor and ion channel evaluation. Both trigeminal ganglia and tongues were

removed, post-fixed in 4% PFA, and cryo-protected in sucrose gradient (20%-50%, 4

oC). Serial sections of frozen trigeminal ganglia (10 μm) and tongue (20 μm) were cut

on a cryostat and thaw-mounted on gelatin-coated slides for processing.

Immunohistochemistry for sensory receptors and proliferation

After sectioning, trigeminal ganglia and tongue sections were briefly rinsed in

PBS, incubated in goat serum (5% in PBS with 0.1% Triton X-100) for 1 hr, then

incubated overnight in the primary antibody. Trigeminal ganglia were stained for PAR-

2 (goat anti-PAR2, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA), TRPV1 (rabbit

anti-TRPV1, 1:400; Fisher scientific, Pittsburg, PA), and TRPA1 (rabbit anti-TRPA1,




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1:200; Abcam, Cambridge, MA). Tongue sections were stained for Ki67 (rabbit anti-

Ki67, 1:400; Abcam, Cambridge, MA), a nuclear protein employed to evaluate

proliferation. After incubation in primary antibody, sections were rinsed in PBS three

times for 10 min each and then incubated in the FITC-AffiniPure goat anti-rabbit

secondary antibody (1:400; Jackson ImmunoResearch Laboratories, West Grove, PA)

for 1 hour at room temperature. Image analysis was performed using NIH Image J

software. The area of staining was outlined and pixel density within the selected area

was then measured and divided by the total area. Data were collected from 4 randomly

selected sections from a minimum of 5 animals per treatment group.

ELISA measurement for cytokines

Plasma IL-6 and TNF-alpha were measured using an ELISA kit from

eBioscience, Inc. (San Diego, CA). Plasma and fat leptin were measured using the

mouse leptin Quantikine ELISA kit (R&D system, Minneapolis, MN). The optical

density of the standards and samples was read at 450 nm wavelength using a Model 680

Microplate Reader (Bio-Rad Laboratories, Inc., Hercules, CA). All samples were run in

triplicate.

Statistical analysis

The statistics software SigmaPlot for Windows (version 11.0) was used to

perform all data analysis. Student’s t –test or Mann-Whitney U test was used to

compare mean or median for the two groups. Repeated-measures ANOVA with one

within-subject factor (time) and one between-subject factor (treatment) followed by

Holm-Sidak post-hoc tests were used to compare the effect of different treatments




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overtime. Simple linear regression was used to examine the correlation of cytokines

with changes in body weight, gnawing time, and tumor size. In the tongue model, three

mice in the control group were euthanized at day 22 due to advanced cancer and severe

cachexia. To best model the trend over time, missing values due to death were treated

using the last observation carried forward method for data analysis and figure

presentation. To make sure our results are not biased by this method, we also analyzed

our data by including the missing values, and found the general conclusions and

statistical significance were not affected. p <.05 was considered statistically significant.

Results are presented as mean ± S.E.M.

Results

NGF mRNA and protein levels were significantly elevated in oral cancer

Tissue biopsies from 14 oral SCC patients showed strong NGF

immunoreactivity (Fig. 1A) whereas the normal oral epithelium from the same patients

showed extremely low NGF labeling (Fig. 1B). NGF mRNA in SCC tumors was

approximately 8.9 times higher than in normal oral tissues (Fig. 1C). NGF protein

concentration was also compared between HSC-3 and NOK cells. Both cell lysate and

supernatant of HSC-3 cells contained much higher NGF levels than those of NOKs

(89.2±3.9 vs. 21.3± 6.6 pg/ml in lysate; 56.6± 3.7 vs. 20.2±5.9 pg/ml in supernatant,

respectively) (Fig. 1D). Even greater elevation of NGF protein was found in cancer

tissues collected from the mouse paw cancer model (30.3±5.1 vs. 4.7±2.0 pg/mg,

respectively) (Fig. 1E).




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Anti-NGF reduced proliferation in culture and in vivo in the tongue

In HSC-3 cell culture, anti-NGF treatment significantly decreased cell

proliferation at 24 (p<.01), 48 (p<.05), and 72 hours (p<.01) after treatment when

compared to the corresponding control group (Fig. 2A). In the tongue tumor mouse

model, anti-NGF treatment reduced tumor volume to about half of that in the control

group (p<.05) (Fig. 2C). We confirmed that the decrease in tumor volume correlated

with a decrease in cancer cell proliferation through quantification of Ki-67 positive cells

in tongue tumors (P<.05) (Fig. 2D and E). In the paw-cancer model, however, all tumor

groups had significantly larger right paw size than the group without tumor (P<.001). In

the anti-NGF treated group, a statistically insignificant trend of tumor volume reduction

was found (Fig. 2B).

Anti-NGF attenuated nociception in the cancer model animals

Paw model: Injection of anti-NGF into the mouse paw tumor reversed tactile

allodynia by an average of 25% relative to that seen in both tumor + PBS (p<.001) and

tumor + CL anti-NGF groups (p<.001), throughout the observation period. Starting on

PID 4, implantation of SCC cells into the right hind paws produced significant

mechanical allodynia (Fig. 3A) consistent with our previous report (28). In naïve mice,

anti-NGF treatment did not cause a hypoalgesic effect (P = .53, Fig. 3A). Both tumor +

PBS and tumor + CL-anti-NGF mice showed a decrease of the same magnitude in paw

withdrawal threshold compared to naïve + anti-NGF mice.

Tongue model: In the tongue-cancer model, all mice developed visible tumor

masses by post-inoculation day 7. Anti-NGF treatment completely abolished the




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progressive increase in gnaw-time caused by cancer at day 21 (p<.05), day 23 (p<.01),

and day 27 (p<.01) (Fig. 3B). Tumor size did not correlate positively with gnaw-time (p

= .35).

Anti-NGF reduced TRPV1, TRPA1, and PAR-2 labeling

Anti-NGF treatment led to a 26% reduction in TRPV1 labeling (P<.05), a 52%

reduction in TRPA1 labeling (P<.001), and a 15% reduction in PAR-2 labeling in

trigeminal ganglion cells (P<.05) (Fig. 3C and D).

Anti-NGF prevented cancer-induced weight loss

Paw- tumor groups treated with anti-NGF did not develop significant weight

loss, whereas both tumor + PBS mice lost more than 5% of their baseline body-mass

starting at the beginning of the third week (Fig. 4A). Unexpectedly, naïve mice that

were treated only with anti-NGF also lost more than 5% body-mass relative to their

baseline at the beginning of the third week (Fig. 4A). At days 18 and 21, anti-NGF

treated tumor mice were significantly heavier than tumor + PBS and naïve + anti-NGF

mice. In the tongue cancer model, by the end of the experiment, the anti-NGF treated

mice retained their original body-mass, whereas the control mice lost 10% of their

body-mass. Significant differences in percent weight change were found at days 17

(p<.05), 21 (p<.001), 23 (p<.01), and 27 (p<.01) (Fig. 4B). The transient body-mass

loss immediately following cancer cell inoculation (Fig. 4B) can be attributed to

attenuated feeding secondary the transient tongue trauma.




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Anti-NGF modulated cytokine levels

Mice treated with anti-NGF exhibited 50% lower plasma TNF-� and IL-6 (Fig.

5A and B) and 3-4 times higher leptin levels in plasma as well as in adipose tissue (the

main source of leptin) (Fig. 5C and D).

Cytokines correlated positively with tumor size, nociception, and weight-

loss

In the tongue-tumor mouse models, IL-6 was positively correlated with tumor

size (R=0.5, p<.05), gnaw-time (R=0.5, p<.05), and weight loss (R=0.6, p<.05) (Fig.

6A, B, and C). TNF-� was positively correlated with weight loss (R=0.8, p<.001) (Fig.

6D) but not with tumor size (P = .16) or gnaw-time (P = .10). Plasma and adipose leptin

concentrations were both inversely correlated with body-mass loss (R=-0.6, p<.05; R=-

0.5, p<.05, respectively) (Fig. 6E and F) but not significantly correlated with tumor size

(P = .1 and P = .86, respectively) or gnaw-time (P = .26 and P = .12).

Discussion

Our results demonstrate that NGF affects progression of oral SCC as well as

pain and cachexia associated with this cancer in patients as well as in mouse models. It

does so, in part by increasing TNF-� and IL-6, by decreasing leptin, and by increasing

expression levels of nociceptive sensory receptors. We demonstrated that NGF

production increased in the orthotopic model of human oral SCC in mice as well as in

oral SCC cell culture. Moreover, NGF blockade with antibodies reduced tumor

proliferation, nociception, and loss of body mass. In summary, NGF blockade: 1)




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yielded lower levels of TNF-� and IL-6, 2) up-regulated leptin, and 3) down-regulated

the nociceptive receptors TRPV1, TRPA1, and PAR-2 in trigeminal ganglia.

The effect of anti-NGF on tumor proliferation varies with tumor type, tumor

location and route of administration. In the current study we evaluated the effect of anti-

NGF on tumor growth in both a paw and a tongue model. For the paw model we

administered the anti-NGF locally in a manner similar to the approach employed by

Adriaenssens et al. in their study of the effect of anti-NGF in a breast cancer model

(14). For the tongue model we administered anti-NGF systemically to preclude the

possibility that repeated injection into the tongue would affect feeding behavior and

maintenance of body mass.

NGF is tumor-promoting for a variety of cancers and anti-NGF has been shown

to reduce tumor proliferation. Andriaenssens et al. demonstrated that locally injected

anti-NGF (12.5μg) decreased tumor size in a breast cancer model (14). However, we

found that local injection of anti-NGF (12.5μg) in a paw model did not have a

significant effect on tumor growth. In our tongue cancer model, anti-NGF significantly

decreased tumor size in animals given a larger systemic dose. The suppressive effect of

NGF on cancer proliferation in-vivo is confirmed by an anti-Ki-67 assay in the tongue

cancer tissue sections and corroborated with an in-vitro proliferation assay.

NGF promotes oral cancer progression in part through a mechanism that

involves IL-6. We investigated whether NGF could exert its effect by modifying

cytokine levels and demonstrated that anti-NGF reduces IL-6. We also found that IL-6

was positively correlated with tumor size. This effect is supported by the clinical

finding that patients with advanced oral cancer exhibit increased IL-6. IL-6 expression




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also correlates with poor prognosis (29, 30). Furthermore, IL-6 activates the

transcription factor NF-kappaB and the STAT3 signal transduction pathway, which in

turn regulate the expression of genes controlling cell proliferation and apoptosis in oral

cancer (29). Additional mechanisms are possible. For example, NGF could also

promote progression by interacting with its receptors TrkA and P75 in both an autocrine

and paracrine manner (31). NGF may also exert its effect on tumor progression by

promoting angiogenesis. NGF has been shown to play a role in angiogenesis in ovarian

and breast cancer (14, 32, 33), and NGF inhibition strongly reduces angiogenesis and

tumor development in mice (14).

Previous studies demonstrated that treatment with anti-NGF has a strong anti-

nociceptive effect in animal models of bone cancer (16, 17). Here we present evidence

that anti-NGF also exhibits a strong anti-nociceptive effect in mice with soft-tissue

cancers. One of the pathways by which NGF induces pain and hyperalgesia entails

modulation of expression and function of nociceptive receptors and sensory ion

channels. For example, the transient receptor potential vanilloid-1 (TRPV1) channel is

known to play a role in cancer pain (3). TRPA1 sensory channels have also been

recently reported to play important roles in orofacial pain (34) and PAR-2 is involved in

animal models of oral cancer pain (25). NGF up-regulates TRPV1 and TRPA1 in

several painful states (11, 34, 35), but a link between NGF and PAR-2 is less certain. In

this study we found that NGF not only modulates TRPV1 and TRPA1 expression, but

also up-regulates PAR-2 expression. Most importantly, anti-NGF reduced TRPA1 by

almost 50%. We infer from this finding that TRPA1 might play an important role in

mediating oral cancer pain.




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In agreement with previous findings (16, 17), we conclude that the anti-

nociceptive effect of anti-NGF does not stem from a reduction in tumor proliferation

alone. First, in the mouse paw model, we did not observe a significant reduction in

tumor size following anti-NGF administration but a significant anti-nociceptive effect

was found. Second, in the tongue cancer model, no correlation was found between

cancer size and gnaw-time. Third, anti-NGF reduced expression of nociceptive

receptors TRPV1, TRPA1, and PAR-2 in trigeminal ganglion cells. This finding

demonstrates that anti-NGF reduces pain at least in part by decreasing nociception

transduction and signaling via these receptors.

Perhaps the most novel finding of our study is that NGF is associated with

cancer-induced cachexia. The unexpected finding that anti-NGF given to naïve mice led

to reduced body mass leaves open the possibility that that a basal level of NGF is

necessary for maintenance of normal body mass. If basal NGF is too low, body mass is

lost. However, when NGF is abnormally elevated, cachexia results. For example, in

animal models of arthritis, NGF levels are elevated and anti-NGF blocks loss of body

mass. These findings support the conclusion that elevated levels of NGF contribute to

arthritis-induced cachexia (36). The manner by which NGF exerts its effect on body

mass regulation is not entirely clear. A variety of cytokines linked to inflammation,

including TNF-�, IL-6, and leptin have been proposed to play a role in body mass

regulation and cachexia (2, 22). TNF-� and IL-6 have been proposed to induce cancer

cachexia through inflammation, altered metabolism, appetite suppression, and enhanced

lipolysis and proteolysis (23). Elevated IL-6 and TNF-� levels have been found in

cachectic cancer patients (22). Both of these cytokines correlate inversely with body




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mass index (BMI) in patients with gastrointestinal cancer (37, 38). Leptin is a cytokine

that is produced mainly by adipocytes (23, 39). This cytokine regulates fat mass by

decreasing the level of neuropeptide Y in the hypothalamus and increasing resting

energy expenditure (23). These changes result in reduced food intake. The relationship

between leptin and cancer cachexia is equivocal. Serum leptin is reduced in cachectic

patients with cancers of the digestive organs (37) and ovaries (40). However, leptin

levels are higher than normal in breast cancer and prostate cancer patients (41, 42). In

patients with oral cancer, reduced leptin levels are accompanied by decreased body

mass (38, 43, 44). Our results suggest that TNF-�, IL-6 and leptin all contribute to oral

cancer-induced loss of body mass. Circulating levels of these molecules can be

influenced by targeting and manipulating NGF levels. Plasma levels of leptin correlate

with levels in adipose tissue. Since plasma leptin is proportional to levels in fat stores, it

is not surprising that untreated cancer mice have decreased plasma leptin as a

consequence of loss of fat mass. However, the ability of adipocytes to produce leptin

might have also been reduced in untreated cancer mice. NGF is known to be secreted

from both murine and human adipocytes in cell culture (45, 46), and fat cells express

the high and low affinity NGF receptors TrkA and P75 (45). Therefore, NGF might act

directly on adipocytes to modulate leptin release. Further studies are needed to elucidate

the mechanism through which NGF regulates body mass and cachexia.

Patients with advanced cancer often suffer from pain and loss of body-mass.

Despite the general consensus that tumor progression, pain, cachexia, and other cancer-

related symptoms result from production and dysregulation of pro-inflammatory

cytokines (1, 8), no studies have been carried out to investigate cancer-related




21

symptoms in parallel. Such an approach might allow us to identify cellular and

molecular mediators common to cancer-related symptoms for a specific tumor type.

We have identified NGF as a key endogenous factor in the panoply of molecules

involved in the orchestration of cancer-related inflammation. NGF might be a molecule

common to the mechanisms responsible for clinically distinctive cancer symptoms such

as pain and cachexia. By suppressing pro-inflammatory cytokines and promoting anti-

inflammatory cytokines, anti-NGF could potentially correct the altered cytokine balance

observed in those who suffer from cancer. Inhibiting chronic inflammation by

neutralizing NGF might be a novel and effective approach to treat pain and cachexia

associated with certain cancers.




22

Acknowledgements: This work was funded by NIH/NIDCR R21 DE018561




23

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Legends for Figures

Figure 1. NGF is elevated in human cancer and mouse models of oral cancer. A.

Immunohistochemistry shows strong NGF immunoreactivity in a representative tissue

biopsy from an oral cancer patient. B. In contrast, normal oral epithelium shows faint

NGF immunoreactivity. C. RT-PCR quantification of NGF mRNA in oral SCC biopsies

shows a large increase over normal tissue. D. NGF protein concentrations in human oral

cancer (HSC-3) cell culture lysate and supernatant are much higher than normal oral

keratinocytes (NOK). E. NGF protein is much higher in the tumor-bearing paw. Scale

bar = 100 μm. *p < .05, **p < .01, ***p < .001 vs. control. Two-tailed Student’s t-test

was used for data analysis.

Figure 2. Anti-NGF therapy decreased cancer proliferation in human cancer cells and

mouse models. A. Anti-NGF decreased cell proliferation rate of cultured HSC-3 cells at

24, 48, and 72 hours. B. Anti-NGF treatment did not affect tumor size in the mouse paw

model. All the tumor groups had significantly larger paw volumes compared to naïve

mice (p<.001). C. Anti-NGF treatment greatly reduced tongue tumor size. D. Ki-67

immunointensity was decreased in a representative tongue tumor following anti-NGF

treatment. E. Quantification of Ki-67 positive cells in total DAPI positive cells showed

significantly less Ki-67 activity following anti-NGF treatment. Horizontal scale bar =

100 μm.*p < .05, **p < .01, ***p < .001; in figure B p < .001 Naïve + anti-NGF vs. all

other three treatment; CL, contralateral paw. Two-tailed Student’s t-test or Mann-

Whitney U test was utilized in figures A, C, E; two-way ANOVA multiple comparisons

was used in figure B.




28

Figure 3. Anti-NGF therapy decreased cancer-induced nociception. A. Anti-NGF

treatment greatly attenuated mechanical allodynia in cancer-bearing paws of mice, but

had no effect on basal mechanical sensitivity. Two-way repeated-measures ANOVA

showed a significant treatment effect (F=511.677, p<.001), and a time × treatment

interaction effect (F=1.661, p<.05); the anti-NGF group was significantly different from

all other groups (p<.001). B. In the tongue cancer model, two-way repeated-measures

ANOVA showed a significant main effect of treatment (F=10.16, p<.001) with a

significant treatment × time interaction (F=2.29, p<.05). Mice treated with anti-NGF

do not show the escalation in gnaw-time but shown by the control mice starting post-

cancer inoculation day 21. C. Immunohistochemistry of a representative trigeminal

ganglion from an anti-NGF treated tongue-cancer mouse shows lower expression of

TRPV1, TRPA1, and PAR-2 receptors. Scale bar = 100 μm. D. Anti-NGF treatment

markedly reduced TRPV1, TRPA1, and PAR-2 immunointensity in trigeminal ganglia

of mice with tongue cancer. *p < .05, **p < .01, ***p < .001

Figure 4. Anti-NGF differentially modulates body weight in normal and cancer-bearing

mice. A. Two-way repeated-measures ANOVA showed a significant main effect of

treatment (F=10.16, p <.001) and time (F=7.63, p<.001). The anti-NGF treated group

was significantly different from tumor + PBS and naïve + anti-NGF (p<.001), but was

not different from tumor + CL-anti-NGF group (p=.09). Vehicle-controlled tumor mice

had significant weight loss from their baseline at day 18 (a). In contrast, no weight loss

is shown for anti-NGF treated mice. Note that naïve mice treated with anti-NGF had

significant weight loss at day 18 (b). At Day 18 and 21, Tumor + anti-NGF group had

significantly larger body-mass compared to tumor + PBS or naïve + anti-NGF groups.




29

B. In mice with tongue tumors, two-way repeated-measures ANOVA showed a

significant main effect of treatment (F=11.53, p<0.001) with a significant treatment ×

time interaction effect (F=2.8, p<0.01). Anti-NGF treatment prevented weight loss

starting at day 17. *p < .05, **p < .01, ***p < .001, #p < .05 tumor + anti-NGF vs.

tumor + PBS, ##p < .01 tumor + anti-NGF vs. tumor + PBS, ++p < .01 tumor + anti-NGF

vs. naïve +anti-NGF, +++p < .001 tumor + anti-NGF vs. naïve +anti-NGF.

Figure 5. Anti-NGF in tongue cancer mouse models decreased plasma TNF-� (A) and

IL-6 (B), and increased plasma and fat leptin (C & D). *p < .05, **p < .01, ***p < .001

vs. control. Two-tailed Student’s t-test was used for data analysis.

Figure 6. Correlation of cytokines with cancer progression, nociception, and weight

change in the tongue cancer model. A, B, C. Plasma IL-6 is positively correlated with

tumor size, gnaw-time, and weight loss. D. TNF-� is positively correlated with weight

loss. E, F. Plasma and fat leptin are inversely correlated with weight loss. Open circles:

control group. Closed circles: anti-NGF treated group. Simple linear regression was

used to test correlation.




Published OnlineFirst July 12, 2011.Mol Cancer Ther Yi Ye, Dongmin Dang, Jianan Zhang, et al. cachexiaNerve growth factor links oral cancer progression, pain, and

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