From non-invasive to invasive breast cancer
Inspecting the trigger

Cancer researchers are seldom, if ever, islands unto themselves. So while their paths had previously never crossed, a chance observation overlapping two individual areas of focus is bringing Nikki Cheng, Ph.D., of Cancer Biology and Fariba Behbod, Ph.D., of Cancer Prevention together as joint principal investigators on a new RO1 grant from the National Cancer Institute. 

Invasive breast cancer research 

Increased expression of CCR2 and CCL2 promote the transition from DCIS to invasive breast cancer. 

The researchers will take a closer look at whether the unpredictable morphing of ductal carcinoma in situ (DCIS) into full-blown invasive breast cancer is influenced by signaling through a protein called CCR2. Cheng has long studied CCR2 and its family of chemokine receptors, which oversee the recruitment of immune cells during infection or chronic inflammation. DCIS, or the presence of abnormal cells within the breasts’ milk ducts, is Behbod’s research specialty. It comprises roughly 20 percent of approximately 200,000 breast disease diagnoses in the U.S. each year, with affected women undergoing a mastectomy if it’s widespread; or a lumpectomy followed by radiation if it’s localized. 

“In fewer than half of DCIS cases do the abnormal cells escape confinement and turn invasive,” Behbod says. “But it happens through processes and mechanisms we don’t fully understand, and because there isn’t yet a good way to predict which person’s DCIS will or won’t spread, every diagnosis has to be treated like it will.”

Behbod has spent several years refining an animal model by which she hopes to identify, among other things, biomarkers of DCIS progression into invasive mode. Called the mouse intraductal (MIND) model, it involves injecting DCIS cells isolated from the tissue samples of consenting patients directly into the milk ducts of female mice. These mouse tumorgrafts are tracked for up to a year alongside a second model of Behbod’s: cultured cells in the laboratory that mimic the course of DCIS from contained to invasive over 10 to 14 weeks. 

Cheng’s expertise in CCR2 function came about somewhat unintentionally: long interested in how connective tissue cells called fibroblasts, a key part of the tumor microenvironment, influence cancer cell behavior, it struck her one day that CCL2 –  a protein secreted by fibroblasts – was “acting odd” in cultured breast cancer cells. “But I didn’t know why,” she says of this behavior. “So I looked at the available literature and discovered that CCL2 binds to and activates CCR2, which is when my lab’s research into chemokine signaling – and how this influences fibroblast interactions with other cell types in the tumor microenvironment – really took off.” She went on to find that CCR2 is overexpressed in breast tumors; it also encourages the survival and invasion of cultured breast cancer cells.

Intrigued by interactions between CCL2 and CCR2 during inflammation and breast cancer progression, Cheng was the first to observe CCR2’s increased signaling activity in a DCIS setting. Aware of Behbod’s MIND model and broad knowledge of this disease, she proposed a collaboration. They’re now investigating their newly-funded grant’s central hypothesis: that active CCR2 signaling promotes DCIS progression to invasive breast cancer, possibly through the bolstering effect such enhanced signaling may have on different cells capable of supporting said progression. 

One exploratory route involves isolating individual epithelial cells from Behbod’s repository of tissue samples, then identifying and injecting those cells with high CCR2 levels into mice. The researchers describe it as “risky, but a method by which to obtain direct evidence of our theory.” Besides the MIND model, they’re combining pre-invasive cancer cells with other cell types, including fibroblasts, and attempting to eavesdrop on any molecular crosstalk foreshadowing invasion. They’ll also genetically manipulate the CCR2 pathway to see how this influences the likelihood of DCIS turning invasive.  

As with most research models, there are limitations. For instance, after as much as a year of tracking, Behbod has yet to see the tumorgrafts in her mice actually morph from what she calls “DCIS-like lesions” into invasive breast cancer. “The mouse version of prolactin doesn’t bind to human tissue, and that’s just one example; it’s probably not the only hormone that behaves differently,” she says. “We’re trying to mitigate this problem by providing the mice with human prolactin. Overall, we need to better mimic the human breast microenvironment and tweak our model’s hormonal milieu, to increase our chances of learning just what triggers DCIS invasion.” 

It’s also likely that multiple factors and molecular pathways are at play in pulling this trigger, with each comprising a worthy research avenue. But in the spirit of starting somewhere, once the kinks of their own approach have been ironed out, Cheng and Behbod hope to have a much clearer idea of both CCR2’s involvement as a biomarker of DCIS progression and its potential utility as a therapeutic target. Perhaps inhibitors could be specifically designed to squelch its hyperactivity; or existing drugs – against chronic inflammatory diseases like rheumatoid arthritis, where CCR2 has a known role – might prove repurposable.

“People have tended to associate CCR2 and the rest of its family with immune reactions, but not tumor survival and invasion,” Cheng observes. “So the correlation we’re proposing here is rather unique. We hope it leads to a solid scientific basis for establishing patient screening tools, based on CCR2 and perhaps additional targets, that better predict and even prevent invasive breast cancer.”

Supporting funds for this research

  • NIH R01CA172764: “Progression of DCIS to Invasive Breast Cancer Through CCR2 Chemokine Signaling”

Relevant publications

  • Valdez KE, Fan F, Smith W, Allred DC, Medina D, Behbod F, “Human primary ductal carcinoma in situ (DCIS) subtype-specific pathology is preserved in a mouse intraductal (MIND) xenograft model.” J Pathology (Dec 2011). 
  • Stacy Hembruff, Iman Jokar, Li Yang, Nikki Cheng, “CCL2 derived from TGF-signaling deficient fibroblasts regulates mammary tumor progression through macrophage dependent and independent mechanisms.” Neoplasia (May 2010).
  •  
  • Wei Bin Fang, Diana Lambert, Iman Jokar, Prasanthi Dendukuri, Nikki Cheng, “CCL2 derived from carcinoma associated fibroblasts enhances mammary carcinoma cell motility and cell growth mediated by p42/44MAPK and Smad3 signaling pathways.” Journal of Biological Chemistry (Aug 2012). 
  • Min Yao, Elaine Yu, Diana Lambert, Vincent Staggs, Fang Fan, Nikki Cheng, "Elevated expression of Chemokine C-C Ligand 2 in stroma is associated with recurrent Basal-like breast cancers." Modern Pathology (2016 Epub ahead of print 10.1038/modpathol 2016.78 PubMed PMID: 27125354).
  • Wei Bin Fang, Min Yao, Gage Brummer, Nabil Alhakamy, Cory Berkland, Nikki Cheng. Targeted gene silencing of CCL2 inhibits triple negative breast cancer progression by blocking cancer stem cell renewal and M2 macrophage recruitment. Oncotarget, (2016 Jun 7. doi: 10.18632/oncotarget.9885. [Epub ahead of print] PubMed PMID: 27283985).
  • Wei Bin Fang, Min Yao, Iman Jokar, Nabil Alhakamy, Cory Berkland, Nikki Cheng, "The CCL2 chemokine is a negative regulator of autophagy and necrosis in luminal B breast cancer cells." Breast Cancer Research and Treatment, (2015, Apr 150(2):309-20 PMID:25744294).
  • Gage Brummer, Diana S. Acevedo, Mike Portsche, Qingting Tina Hu,  Wei Bin Fang, Min Yao, Kyle Warren,  Brandon Zinda, Yan Hong, Fariba Behbod, Nikki Cheng, "CCL2/CCR2 chemokine signaling regulates the transition of ductal carcinoma in situ to invasive breast cancer." (Manuscript in preparation.)
  • Wei Bin Fang, Kyle Warren, Garth Fraga, Li-Ching Huang, Yu Shyr, Lu Xie, Vincent Staggs, Nikki Cheng, "A CCL2 chemokine signaling pathway as a prognostic signature to distinguish breast ductal carcinoma in situ associated with invasive progression." (Manuscript submitted; under revision.)