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STAT

An MUSC blog
Keyword: metastasis

Primary colorectal tumors secrete VEGF-A, inducing CXCL1 and CXCR2-positive myeloid-derived suppressor cell (MDSC) recruitment at distant sites and establishing niches for future metastases, report Medical University of South Carolina (MUSC) investigators in an article published online ahead of print on April 28, 2017 by Cancer Research. Liver-infiltrating MDSCs help bypass immune responses and facilitate tumor cell survival in the new location. This research illuminates mechanisms by which primary tumors contribute to premetastatic niche formation and suggests CXCR2 antagonists may reduce metastasis.

Recent cancer research shows that premetastatic 'niches' form at sites far from the original tumor before new tumors occur. In colorectal cancer (CRC), these supportive microenvironments form in preferred secondary organs, such as the liver and lung, and facilitate the colonization, survival, and growth of metastasizing tumor cells. However, the mechanisms responsible for the formation of these premetastatic 'niches,' including what role(s) the primary tumor may play, are not well understood. It is critical to better understand the mechanics of CRC metastasis, as it is the second leading cause of cancer deaths in the US and patients with advanced cases often die because current treatments for widely metastasized disease are not effective.

MUSC investDr. Raymond N. DuBois, dean of the Medical University of South Carolina College of Medicineigators led by Raymond N. DuBois, M.D., Ph.D., dean of the MUSC College of Medicine and professor of Biochemistry and Molecular Biology, have now illuminated how primary CRC tumors contribute to premetastatic 'niche' formation.

"The idea that some sort of 'priming' needs to take place for metastasis to occur in distant organs - that there is some sort of activity in the future tumor location - is not new. But most research has focused on growth factors, chemokines and pro-inflammatory cytokines. There hasn't been much work looking at immune cell activity in distant organs prior to metastasis," explains DuBois. "We knew that the type and density of immune cells in the primary tumor plays a role in progression. For example, when more immature myeloid cells are present in the tumor, it becomes resistant to immune attack. But we didn't know what to expect in a metastatic model."

To explore this area, the team first evaluated whether the presence of a primary tumor affected immune cell profiles in premetastatic liver and lung tissues of mice. They found that the presence of a primary cecal tumor caused MDSCs to begin infiltrating the liver before metastasis began. Working backward from this finding, they used a series of experiments to reveal the chain of events that led up to MDSC infiltration.

Because CXCR2 is essential for drawing MDSCs out of the bloodstream and toward CRC tumors and colonic mucosa, the team began looking for CXCR2 and its ligands (CXCL1, CXCL2, and CXCL5) in mouse liver tissue. The team not only found that the ligand, CXCL1, attracted MDSCs from the bloodstream into premetastatic liver tissue, but also that administering a CXCR2 antagonist inhibited CXCL1 chemotaxis. This demonstrated that CXCR2 is required for CXCL1 to induce MDSC liver infiltration. In other words, the CXCL1-CXCR2 axis is required to recruit MDSCs to the liver. Importantly, they also found that liver- infiltrating MDSCs secrete factors that promote cancer cell survival and metastatic tumor formation without invoking the innate and adaptive immune responses.

Next, because VEGF is known to induce CXCL1 expression in lung cancer, the research team examined whether VEGF secreted by CRC tumors also regulated CXCL1 expression. Their results demonstrated that VEGF-A secretion by primary CRC tumor cells stimulates macrophages to produce CXCL1. Interestingly, although VEGF-A knockdown inhibited liver metastasis, it did not affect the growth of the primary tumor.

"We did not expect to find that a primary tumor could affect a distant organ before any of the cancer cells arrived on site," says DuBois. "We were surprised to see these changes before a single metastatic cell took up residence."

Together, these studies reveal that VEGF-A secreted by the primary CRC tumor stimulates macrophages to produce CXCR1, which recruits CXCR2-expressing MDSCs from the bloodstream into healthy liver tissue. The MDSCs then create a premetastatic 'niche' or micro-environment where cancer cells can grow to form new tumors. These results demonstrate for the first time that cells in the primary tumor contribute to forming distant pre-metastatic 'niches' which facilitate the spread of disease.

"Now that we know the primary tumor puts things in motion remotely prior to metastasis, we should be able to inhibit this process and have a positive impact on survival," explains DuBois. "We now know which molecules and immune cells are involved and that if we disrupt the CXCL1-CXCR2 axis we can possibly reduce the spread of disease. Both antibodies and small molecules can inhibit this pathway, but they have not yet been optimized. I hope these findings will speed up the development of inhibitors of the CXCR2 pathway."

Immunofluorescence analysis to detect the expression and localization of Vps34 and Beclin-1 in cathepsin B overexpressing mouse mammary epithelial (CTSB/OE cells) treated -/+ TGF-beta for 7 days.

 

 

 

 

 

 

 

 

 

Summary: In an article published online in Nature Cell Biology on July 11, 2016, investigators at the Medical University of South Carolina report preclinical findings suggesting that disabled 2 (Dab2) serves as a molecular switch that regulates whether a tumor cell undergoes autophagy or apoptosis. Maintaining Dab2 by inhibiting cathepsin B could prevent tumor cell survival by blocking autophagy and promoting cell death. These insights provide important information for maximizing the efficacy of existing chemotherapeutic agents.

The results of preclinical studies by investigators at the Medical University of South Carolina (MUSC) reported in an article published online on July 11, 2016 in Nature Cell Biology (doi: 10.1038/ncb3388) demonstrate that disabled 2 (Dab2) serves as a molecular switch that regulates whether a tumor cell undergoes autophagy or apoptosis.

While expression of Dab2—an endocytic adaptor and tumor suppressor—is known to occur during transforming growth factor-beta (TGF-beta)–mediated epithelial-mesenchymal transition (EMT), the mechanisms by which it regulates apoptosis were, until now, poorly understood.

Exploring the pathways by which Dab2 is degraded and the effects of maintaining Dab2 levels reveals its pivotal role in preventing tumor cell survival by blocking autophagy and promoting cell death. These insights provide important information for maximizing the efficacy of existing chemotherapeutic agents.

TGF-beta induces EMT—a process by which cells transform from a polarized epithelial phenotype to a fibroblastic or mesenchymal one. Dab2 is expressed during TGF-beta–mediated EMT. While EMT is essential for normal cellular growth and homeostasis, it is abnormally activated in tumor cells and contributes to their chemo-resistance and metastasis.

TGF-beta has also been reported to regulate autophagy, which, in established tumors, ensures tumor cell survival through times of stress, as for example during chemotherapy. In other words, autophagy supports the chemo-resistance, growth, and metastasis of tumor cells.

Researchers focused on the Dab2 protein after noticing that, in cells treated with TGF-beta, Dab2 levels rose over the initial 24-48 hours as they went through EMT but then fell with continued TGF-beta treatment. By day 7, the tumor cells had transitioned to a morphological state suggestive of either autophagy or apoptosis. Furthermore, the mesenchymal markers N-cadherin and vimentin, which like Dab2 initially rose during EMT, began to decline with longer exposure to TGF-beta.

"This was an unexpected finding that we followed,” explains senior author Philip Howe, Ph.D., Professor and Chair of Biochemistry and Molecular Biology and Hans and Helen Koebig Chair in Clinical Oncology at MUSC. “We knew that if you give cells TGF-beta they go through EMT, and we knew you needed Dab2 for TGF-beta–mediated EMT. But, when we kept adding TGF-beta for more sustained periods (after EMT took place), cells took on a different morphology and we noticed a loss of Dab2. We investigated this loss of Dab2 and discovered that it was being cleaved and that the cells were undergoing autophagy. Upon sustained TGF-beta treatment, the cells had lost their mesenchymal phenotype they'd gained in EMT and entered into an autophagic state.”

The team began to explore how prolonged TGF-beta treatment led to loss of Dab2 and the mesenchymal phenotype.

First, they found that longer TGF-beta exposure significantly increased cathepsin B (CTSB) expression and promoted its co-localization with Dab2. The team then not only demonstrated that CTSB is responsible for cleaving Dab2 but also that it recognizes the cleavage site by the flanking amino acids Val499 and Gly500. Thus, while an unaltered Dab2 sequence (Leu-Val-Gly-Leu) was degraded by CTSB, it did not cleave a mutant Dab2 sequence (Leu-Val-Leu).

Second, findings showed that, after 7 days, continuous TGF-beta treatment induced autophagy and down-regulated markers of apoptosis. This was particularly notable because these conditions promote tumor cell chemo-resistance and metastasis.

Third, they found that CTSB inhibition or expression of a mutant Dab2 without the CTSB cleavage site (i.e., the Leu-Val-Leu mutant) led to time-dependent increases in pro-apoptotic markers. When TGF-beta was withdrawn, cells in which Dab2 had been preserved underwent cell death. This series of experiments show not only how Dab2 is modulated by CTSB but also that it serves as a switch for regulating TGF-b–induced autophagy and apoptosis.

Another series of experiments were undertaken to clarify exactly how Dab2 functions to prevent autophagy and promote apoptosis. These findings show that Dab2 inhibits TGF-beta–induced autophagy by blocking the Vps-Beclin-1 interaction and promotes apoptosis by attenuating ERK-Bim interactions.

Finally, the team used the chemotherapeutic agent doxorubicin (DOXO) to determine whether the role of Dab2 in inhibiting autophagy might affect tumor cell chemo-sensitivity. They found that cells in which CTSB was overexpressed had increased survival in the presence of DOXO. However, cells with high Dab2 levels due to CTSB inhibition or expression of the CTSB-resistant Dab2 mutant were more chemo-sensitive and underwent apoptotic changes. Thus, Dab2 was shown to promote chemotherapeutic drug–induced cell death by attenuating drug-induced autophagy. In vivo tumor studies in mice further found that Dab2 both enhanced DOXO-mediated cell death and attenuated tumor cell metastasis.   

These direct insights into molecular mechanisms supporting tumor cell survival and death are crucial for maximizing the effectiveness of existing chemotherapeutic agents. "This is important because there aren't a whole lot of drugs out there," explains Howe. "Most of what we use today has been around for 20 or 30 years because of a lack of investment in basic science." 

The team's next steps are to investigate in vivo models for combination therapies using DOXO and a CTSB inhibitor to further illuminate the potential for targeting Dab2 as a means of reducing tumor recurrence and metastasis.

Image Caption: In the absence of Disabled-2 (Dab2), Vps34/Beclin-1 Interactions are maintained. Immunofluorescence analysis to detect the expression and localization of Vps34 and Beclin-1 in cathepsin B overexpressing mouse mammary epithelial (CTSB/OE cells) treated -/+ TGF-beta for 7 days. Photos were taken by confocal microscope. Scale bars, 10 mm. The data show that in the absence of Dab2, due to CTSB overexpression, Vsp34/Beclin-1 interactions are maintained and autophagy is initiated. Adapted from a figure originally published in an article by Jiang Y, Woosley AN, Sivalingam N, Natarajan S, and Howe PH in Nature Cell Biology (doi: 10.1038/ncb3388).

 

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