Stiffer Matrix in Fat Tissue May Promote Breast Cancer Risk

Alexander M. Castellino, PhD

August 25, 2015

The link between obesity and increased risk for cancer has been well documented, especially for breast cancer in women. Although biochemical changes have been proposed to explain the mechanism behind the way obesity increases the risk for cancer, a new study shows that the extracellular matrix (ECM) — the space that surrounds cells — of the adipose tissue is stiffer in obesity and is characterized by ECM components typically associated with the tumor phenotype.

These observations were published in the August 19 issue of Science Translational Medicine by researchers from Cornell University, Ithaca, New York, and were based on experiments first from a genetic mouse model of obesity and then, separately, in a diet-induced mouse model. The research group, led by Claudia Fischbach, PhD, associate professor of biomedical engineering, also showed that the stiff ECM promotes proliferation of breast cancer cells and can influence the behavior of premalignant cells.

For the genetic mouse model of obesity, researchers used female mice lacking the leptin gene associated with satiety; these animals "are morbidly obese compared with age-match wild-type controls." In the diet-induced mouse model, obesity was achieved by feeding the animals a high-fat diet for 10 weeks.

"We wanted to be certain that our results were not interpreted as a leptin-dependent mechanism and that is why we replicated most of our observations in the diet-induced mouse model, which is more tedious to do," corresponding author and principal investigator Dr Fischbach told Medscape Medical News.

These observations also have implications for breast reconstructive surgery where implanting adipose tissue is done often, she added.

"Although this new insight adds to the emerging complexity by which obesity fuels tumor growth and progression, it also may help to integrate the various mechanisms that link obesity-related risk to malignancy — thus providing a basis for clinical interventions," experts write in an accompanying commentary.

The editorialists are Lisa M. Arendt, DVM, PhD, from the Department of Comparative Biosciences, University of Wisconsin–Madison, and Charlotte Kuperwasser, PhD, from the Developmental, Molecular, and Chemical Biology Department at the Tufts University School of Medicine, Boston.

The research conducted in Dr Fischbach's laboratory was multitiered. The group first looked at how ECM is remodeled in obesity. The team then determined how the remodeled ECM could influence proliferation of breast cancer cells. Finally, the researchers showed how the ECM seen in animal models of obesity was replicated in both tumor-free and tumor tissue of obese women with breast cancer.

ECM Remodeling in Obesity

In defining ECM remodeling, Dr Fischbach's research team showed that, compared with wild-type or lean control subjects, changes in the ECM of adipose breast tissue in obese animals were not only due to increased levels of ECM components, but also the manner in which some of them were deposited. Histologic staining showed increased levels of interstitial alpha-smooth muscle actin (alpha-SMA) — a marker of myofibroblasts — and fibronectin. Structural testing using "second harmonic generation" showed that collagen deposited between the cells was structurally different in obese animals.

Another feature of the ECM of adipose mammary tissue from obese animals was the type of fibronectin deposited by adipose stromal cells (ASCs) surrounding the ECM. The fibronectin was partially unfolded and contributed to the stiffness of the ECM, which was reminiscent of matrix rigidity observed by others to be associated with malignancy.

Obesity promotes stiffening of the extracellular matrix in breast tissue. Second harmonic imaging of murine breast tissue demonstrates curly collagen fibers in normal-weight mice but increasingly straight fibers in obese animals. Images are pseudo-colored to show collagen fibers in magenta and adipocytes and inflammatory cells in green (Courtesy of the Fischbach lab).

The cells that contributed to these profibrotic changes in the tissue microenvironment were myofibroblasts, the researchers showed. ASCs from mammary tissue of obese animals had increased levels of alpha-SMA-positive myofibroblasts, and showed an increased capacity for proliferation. Indeed, obesity-associated ASCs were capable of assembling "thicker ECMs enriched with collagen and fibronectin."

The "interstitial mechanics" of breast tissue from obese and normal animals using atomic force microscopy nanoindentation further confirmed that "obesity affects the mechanical properties of the ECM deposited by the ASCs, which may ultimately lead to interstitial stiffening."

A Stiffer ECM May Induce a Malignant Phenotype

Can a stiffer ECM from adipose tissue sustain tumorigenesis? To prove that it can, the researchers showed that a decellularized ECM from obesity-associated ASCs was capable of promoting the proliferation of human breast cancer cells more so than a decellularized ECM from normal control subjects. Further, they showed that this stiff ECM was capable of altering the contractility of the cells relative to a normal ECM.

"Usage of cell surface receptors such as integrins is different in stiffer versus a less stiff matrix, which ultimately contributes to increased tumor cell migration and invasion," Dr Fischbach told Medscape Medical News.

The decellularized, stiffer ECM from ASCs derived from obese animals also promoted the proliferation and disorganized growth of premalignant cells, which were also shown to migrate along the ECM fibers away from their original position.

All these observations were suggestive that the stiffer ECM had the capability for cellular proliferation and cellular contractility, which is associated with invasiveness and migration of tumor cells.

Clinical Relevance

The clinical relevance of these findings was explored in a histochemical analysis of tumor-free breast tissue from normal, overweight, and obese patients.

The analyses revealed that obese tissues had increased alpha-SMA and fibronectin, and thicker collagen fibers. Alpha-SMA also correlated with inflammation and fibrosis of adipose tissue as determined from histologic signatures compared with those from lean patients.

"Our results demonstrate that obesity leads to the formation of mechanical niches in adipose tissue that mimic characteristics of tumor-associated stroma and promote carcinogenesis by altering mechanotransduction," the researchers write.

"[The] altered ECM not only provides a positive feedback mechanism for further myofibroblast differentiation but can also increase the malignant behavior of mammary epithelial cells," they add.

Important Observations

The observations are important from a standpoint of detection and breast reconstructive surgery, Dr Fischbach explained to Medscape Medical News. Mammograms may not detect the observed small regions of dense tissue in obese women because it is hidden by fat cells, she said.

"[Our] data suggest that local, microscale density changes still occur. Therefore, alternative high-resolution techniques will be necessary to better localize and monitor regions that may stimulate tumorigenesis in obese patients," she states.

The data also have implications for the use of adipose tissue in reconstructive breast surgery.

"The clinical practice of using noncancer adipose tissue in breast reconstructive surgery may need to be reevaluated in obese women with breast cancer," Dr Fischbach told Medscape Medical News.

"Implanting adipose tissue for breast reconstruction following surgery for breast cancer has been controversial with respect to its safety," Dr Fischbach said.

She states: "Thus far, little attention has been given to the body habitus of the patient from whom the ASCs are isolated, yet our data imply that ASCs from obese individuals might promote recurrence of breast cancer relative to ASCs from lean patients."

In their commentary, Drs Arendt and Kuperwasser discuss some of the therapeutic implications for obese women with breast cancer.

The new findings may explain results that have been seen clinically with aromatase inhibitors. In postmenopausal women, estrogen is mainly produced by adipocytes through activity of the enzyme aromatase, they write. This has led to the hypothesis that aromatase inhibitors may be particularly beneficial for obese women in the treatment of estrogen-receptor-positive breast cancer. However, in multiple epidemiologic studies, aromatase inhibitors had similar or reduced benefit compared with that of tamoxifen in obese postmenopausal breast cancer patients, with obese patients potentially demonstrating increased relapse after treatment, they point out.

They also note that increased circulating levels of insulin suggest that metformin — currently used in patients with diabetes — holds promise as adjuvant therapy. Large clinical trials evaluating the efficacy of metformin in obesity-associated breast cancer are ongoing, they indicate.

After treatment, weight loss is a strong clinical recommendation for obese women with breast cancer. Although Dr Fischbach and her team showed that calorie restriction in obese animals was associated with a reduced number of alpha-SMA-positive myofibrils in the mammary glands, effects on fibronectin and ECM remodeling were less clear.

"Identifying how weight loss contributes to changes in the ECM and ultimately breast cancer risk are critical for advising patient care," Drs Arendt and Kuperwasser write.

"Because therapeutics specifically targeted for obese breast cancer patients are lacking, and weight reduction is recommended for breast cancer survivors, further research is necessary to determine whether dietary or exercise interventions or both are effective in the amelioration of ECM changes induced by obesity," they conclude.

The authors, Dr Arendt, and Dr Kuperwasser have disclosed no relevant financial relationships.

Sci Transl Med. 2015;7:301ra130, 301fs34. Abstract, Commentary

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