A CXCL1 Paracrine Network Links Cancer Chemoresistance and Metastasis

Knockdown of CXCL1 and CXCL2 using two independent shRNA hairpins significantly reduces mammary tumor growth in all three models.

“Knockdown of CXCL1 and 2 using two independent shRNA hairpins (Figure S1C–D) significantly reduced mammary tumor growth in all three models (Figures 1C–D, 1G–H and S1E–F)”

A significant reduction in CD11b+Gr1+ myeloid cells is observed in CXCL1/2 knockdown tumors in both models.

“A significant reduction in CD11b+Gr1+ myeloid cells was observed in CXCL1/2 knockdown tumors in both models (Figure S2G–H).”

CXCL1/2 knockdown is associated with reduced metastasis to the lungs.

“This decrease was associated with reduced metastasis in the lungs (Figures 1E–F, 1I–J and S1G).”

CXCL1 and CXCL2 show a similar expression pattern in a combined cohort of 615 primary breast cancers.

“Indeed, CXCL1 and CXCL2 showed a similar expression pattern in a combined cohort of 615 primary breast cancers (Figure 1A).”

CXCL1 and CXCL2 are amplified in 7.5% of primary breast tumors and 19.9% of metastases by FISH.

“Fluorescence in situ hybridization (FISH) of human tissue samples showed that CXCL1 and CXCL2 were amplified in 7.5% of primary breast tumors and in 19.9% of metastases (Figures 1B and S1A).”

LM2 cells show upregulation of CXCL1/2 compared to the parental MDA-MB-231 line.

“LM2 cells showed upregulation of CXCL1/2 compared to the parental line (Figure S1B).”

Lung colonization assays by tail vein injection confirm that the effect of CXCL1/2 depletion on metastasis is not solely due to decreased tumor burden.

“Furthermore, lung colonization assays by tail vein injection of LM2 cells confirmed that the effect of CXCL1/2 depletion on metastasis is not solely a consequence of decreased tumor burden (Figure 1K–N).”

Compared to high CXCL1/2 expression in lung metastatic cell lines, expression of CXCR1, CXCR2 and DARC is negligibly low at RNA and protein levels.

“Compared to the high levels of CXCL1/2 expression in lung metastatic cell lines, the expression of CXCR1, CXCR2 and DARC was negligibly low both at the RNA and protein levels (Figure 2C and Figure S2F and (Muller et al., 2001)).”

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Detailed characterization shows a decrease in CD11b+Ly6G+ granulocytic MDSC population in CXCL1/2 knockdown tumors compared to controls.

“Detailed characterization of the myeloid cells showed a decrease in the CD11b+Ly6G+ granulocytic MDSC population in CXCL1/2 knockdown tumors compared to controls (Figure 2D and S2I).”

Reduction in CXCL1/2 levels in LM2 xenograft and PyMT transplantation models is associated with a significant increase in apoptosis in tumors.

“Reduction in CXCL1/2 levels in the LM2 xenograft and PyMT transplantation models was associated with a significant increase in apoptosis in the tumors (Figure 2A–B).”

Size-matching knockdown tumors to controls yields similar results.

“Similar results were obtained upon size-matching the knockdown tumors to controls (Figure S1H and I).”

Analysis of 67 metastases from breast cancer patients shows a 61% overlap in CXCL1-associated genes between primary breast tumors and metastases.

“Similar analysis of a dataset generated from 67 metastases from breast cancer patients showed a 61% overlap in CXCL1-associated genes between primary breast tumors and metastases (Figure 3B; Tables S3, S4).”

In a qRT-PCR panel of typical NFκB/STAT1 activators, TNF-α was strongly induced in endothelial cells after doxorubicin treatment.

“Among a panel of prototypical activators of the NFκB/STAT1 pathway that we assayed by qRT-PCR, TNF-α was strikingly induced in endothelial cells upon doxorubicin chemotherapy treatment (Figure 6B).”

In addition to CXCL1/2 and S100A8/9, the CXCL1-associated chemokine genes CCL20 and CXCL3 are also highly expressed in response to chemotherapy.

“In addition to CXCL1/2 and S100A8/9, the CXCL1-associated chemokine genes CCL20 and CXCL3 were also highly expressed in response to chemotherapy (Figure S5B).”

When LM2 tumor cells were cultured with conditioned medium from chemotherapy-treated primary endothelial cells or primary bone marrow-derived cells, CXCL1/2 expression rose significantly.

“However, LM2 tumor cells incubated with conditioned media from chemotherapy-treated primary endothelial cells or primary bone marrow-derived cells showed a significant increase in CXCL1/2 expression (Figures 6A and S6B).”

A significant increase in S100A9 expressing cells is observed in breast cancers after chemotherapy treatment.

“Consistent with our experimental models, a significant increase in S100A9 expressing cells was observed in breast cancers after chemotherapy treatment (Figure 5G–H and Table S5).”

There is no discernible increase in senescence in LM2 tumors upon AC chemotherapy treatment.

“Although induction of CXCL chemokines occurs during chemotherapy-induced senescence (Coppe et al., 2008), there was no discernible increase in senescence in LM2 tumors upon AC chemotherapy treatment (Figure S5C–D).”

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Enforced expression of S100A8/9 in LM2 cells rescues the CXCL1/2 knockdown phenotype of reduced tumor growth and metastasis.

“Furthermore, the enforced expression of S100A8/9 in LM2 cells (Figure S3G–H) rescued the CXCL1/2 knockdown phenotype of reduced tumor growth and metastasis (Figures 3H,I and S3I).”

Infliximab, an anti-TNF-α antibody, reduced the recruitment of S100A9-expressing cells in chemotherapy-treated mammary tumors.

“Treatment with anti-TNF-α antibody infliximab reduced recruitment of S100A9-expressing cells in chemotherapy-treated mammary tumors (Figure 6G–H).”

After chemotherapy, histopathological analysis showed strong TNF-α staining in tumor microenvironment cells, particularly lymphatic and blood vessels and fibroblast-rich stroma.

“Importantly, histopathological analysis revealed that cells from the tumor microenvironment specifically lymphatic and blood vessels and fibroblast-rich stroma showed strong TNF-α staining after chemotherapy (Figure 6J and S6D).”

Lymphocytic cells represent a minority of leukocytes infiltrated in immunocompetent PyMT transplanted tumors compared to myeloid components.

“Compared to the myeloid components, lymphocytic cells represented a minority of leukocytes infiltrated in the immunocompetent PyMT transplanted tumors (Table S1).”

TNF-α immunostaining increased significantly in patient samples after neoadjuvant AC chemotherapy.

“Consistent with our hypothesis, a significant increase in TNF-α immunostaining was observed in patient samples after neoadjuvant AC chemotherapy treatment (Figure 6I–K)”

Chemotherapy also induced TNF-α in smooth muscle cells and bone marrow-derived cells.

“TNF-α induction in response to chemotherapy also occurred in smooth muscle and bone marrow derived cells (Figure 6E)”

Purified lung endothelial cells from LM2 tumor-bearing mice given systemic AC chemotherapy showed a tenfold rise in TNF-α expression.

“Moreover, we observed a ten-fold increase in TNF-α expression in purified lung endothelial cells from LM2 tumor bearing mice systemically treated with AC chemotherapy (Figure 6C,D).”

The requirement of S100A8/9 in cell survival is even more evident when tumor-bearing mice are treated with chemotherapeutic agents as an inducer of cell death.

“The requirement of S100A8/9 in cell survival was even more evident when tumor-bearing mice were treated with chemotherapeutic agents as an inducer of cell death (Figure 4B–C and S4A).”

In the authors' experimental models, chemotherapy-induced increases in CXCL1/2 were not attributable to extra copies of the gene locus, as assessed by FISH.

“In our experimental models, enhanced expression of CXCL1/2 in response to chemotherapy was not due to additional amplification of the locus as determined by FISH analysis (Figure S6A).”

CXCL1/2 upregulation in LM2 tumors is also observed with paclitaxel.

“CXCL1/2 upregulation in the LM2 tumors was but was also observed with another commonly used chemotherapeutic agent, paclitaxel (Figure S5A).”

After subsequent rounds of chemotherapy, tumors become refractory as evidenced by reduction in apoptosis and resumed tumor growth.

“However, after subsequent rounds of chemotherapy, tumors became refractory as evidenced by reduction in apoptosis and resumed tumor growth (Figure 5A–B).”

These 43 CXCL1-correlated genes show a predominance of chemokines (40%) and cytokines (21%).

“These genes showed a predominance of chemokines (40%) and cytokines (21%).”

Tumor-bearing mice transplanted with S100A9-/- bone marrow show a marked increase in apoptotic cells in mammary tumors and lung.

“Tumor-bearing mice transplanted with S100A9−/− bone marrow showed a marked increase in the number of apoptotic cells in the mammary tumors and the lung (Figure 4A).”

Similar numbers of CD11b+ and CD68+ myeloid cells are present in tumors and lung nodules from mice transplanted with either S100A9-/- or S100A9+/+ bone marrow cells.

“Similar numbers of CD11b+ and CD68+ myeloid cells were present in tumors and lung nodules from mice transplanted with either S100A9−/− or S100A9+/+ bone marrow cells (Figure S4B and data not shown).”

Analysis of 615 breast tumors from three independent datasets yielded 43 genes that correlate with CXCL1 with a coefficient greater than 0.3.

“Analysis of 615 breast tumors from three independent datasets yielded 43 such genes that correlated with CXCL1 with a coefficient >0.3 (Figure 3A and Table S3).”

Exposing LM2 cells directly to the chemotherapeutic drugs did not raise CXCL1/2 expression.

“Direct treatment of LM2 cells with the chemotherapeutic agents did not induce CXCL1/2 expression (Figure S6B and data not shown).”

Co-culture of cancer cells with bone marrow derived CD11b+Gr1+ cells protects cancer cells from doxorubicin-induced apoptosis.

“Consistent with the survival advantage provided by S100A8/9 in vivo, co-culture of cancer cells with bone marrow derived CD11b+Gr1+ cells protected cancer cells from doxorubicin-induced apoptosis (Figure 4D).”

Using the NFκB pathway inhibitor NBD, the authors found that TNF-α-driven NFκB activation stimulated CXCL1/2 expression in LM2 tumor cells.

“NFκB activation by TNF-α stimulated the expression of CXCL1/2 in LM2 tumor cells, as determined with the use of NFκB pathway inhibitor (Nemo binding domain peptide or NBD) (Figure 6F).”

TNF-α immunostaining increased significantly in LM2 mammary tumors from mice treated with AC chemotherapy.

“and in LM2 mammary tumors in mice treated with AC chemotherapy (Figure S6C).”

Patients with high S100A8/9 in metastatic nodules have significantly shorter overall survival compared to low S100A8/9 (p=0.01).

“Kaplan Meier analysis showed that patients with high S100A8/9 in the metastatic nodules had a significantly shorter overall survival compared to low S100A8/9 (p-value=0.01) (Figure 3K).”

Mammary tumor growth and lung metastasis are significantly reduced in mice transplanted with S100a9-/- bone marrow compared to S100a9+/+ counterpart.

“Mammary tumor growth and lung metastasis were significantly reduced in mice transplanted with S100a9−/− bone marrow compared to the S100a9+/+ counterpart (Figure 3E–G).”

S100A8/9 cause activation of ERK1/2, p38 MAPK, and p70S6K in LM2 cells.

“Using blot arrays that detect phosphorylation of 46 different protein kinases, we found that S100A8/9 cause activation of ERK1/2, p38 MAPK, and p70S6K in LM2 cells (Figures 4E and S4D), as confirmed by immunoblotting (Figures S4E–F).”

The CXCR2 inhibitor alone had partial or no effect, depending on the model.

“As a single agent CXCR2 inhibitor had partial to no effect depending on the model system.”

Mice bearing tumors and treated with AC chemotherapy alone showed reduced tumor growth.

“Tumor-bearing mice treated with AC chemotherapy alone showed a reduction in tumor growth (Figures 7A,B and S7A,B).”

S100A8/9 activate p70S6K, contributing to their pro-survival effect in these cells.

“In addition to activating MAPK pathways (Gebhardt et al., 2006; Ichikawa et al., 2011), we find that S100A8/9 activate p70S6K as contributors to the pro-survival effect of S100A8/9 in these cells.”

Clinically and biologically, metastasis is intricately linked with resistance to chemotherapy.

“Clinically as well as biologically, metastasis is intricately linked with resistance to chemotherapy (Hu et al., 2009; Morris et al., 2009).”

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Cancer cells that overexpress CXCL1 and CXCL2 through transcriptional hyperactivation or 4q21 amplification are primed for survival in metastatic sites.

“Cancer cells that overexpress CXCL1 and 2 by transcriptional hyperactivation or 4q21 amplification are primed for survival in metastatic sites.”

The endothelial-carcinoma-myeloid signaling network provides a mechanism linking chemoresistance and metastasis.

“This network of endothelial-carcinoma-myeloid signaling interactions provides a mechanism linking chemoresistance and metastasis, with opportunities for intervention.”

Increased copy number contributes to higher CXCL1/2 expression in invasive breast tumors and metastases.

“These results suggested that increased copy number contributes to the higher CXCL1/2 expression in invasive breast tumors and metastases.”

A paracrine signaling network involving carcinoma, myeloid and endothelial cells drives both metastasis and chemoresistance in breast cancer.

“We uncovered a network of paracrine signals between carcinoma, myeloid and endothelial cells that drives both processes in breast cancer.”

• Also stated in: § INTRODUCTION · plain

  “We uncovered a paracrine network, with the chemokines CXCL1 and 2 at its core that mediates lung metastasis and chemoresistance in breast cancer.”

CXCL1/2 play a prominent role in breast cancer progression and metastasis.

“We concluded that CXCL1/2 play a prominent role in breast cancer progression and metastasis.”

ERK1/2 and p70S6K signaling mediate the pro-survival effect of S100A8/9 in metastatic cells.

“suggesting that the ERK1/2 and p70S6K signaling mediate the pro-survival effect of S100A8/9 in metastatic cells.”

Successful engraftment of S100a9+/+ or S100a9-/- bone marrow was confirmed with an efficiency greater than 98%.

“After confirming successful engraftment of S100a9+/+ or S100a9−/− bonemarrow with an efficiency of >98% (Figure S3C)”

The authors' findings suggest that at least some chemotherapy drugs have a self-defeating effect by causing stromal release of pro-inflammatory cytokines such as TNF-α.

“Our findings suggest that a self-defeating consequence of at least some chemotherapy drugs is the release of potent pro-inflammatory cytokines such as TNF-α from stromal sources.”

Higher apoptotic rates in the S100A9-/- group are not due to defective accumulation of myeloid cells.

“This result ruled out the possibility that higher apoptotic rates in the S100A9−/− group were due to a defective accumulation of myeloid cells (Hiratsuka et al., 2008).”

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S100A8/9 mediate the pro-metastatic effect of CXCL1/2 in these models.

“Together, these results suggest that S100A8/9 mediate the pro-metastatic effect of CXCL1/2 in our models.”

Chemotherapy activates a burst of paracrine factors including the cancer cell survival axis CXCL1/2-S100A8/9 that selects for cancer cells that can resist chemotherapy.

“Together, these results suggest that chemotherapy activates a burst of paracrine factors including the cancer cell survival axis CXCL1/2-S100A8/9 that selects for cancer cells that can resist chemotherapy.”

TNF-α release is a general stromal response to chemotherapy across different cell types.

“suggesting that TNF-α release is a general response to chemotherapy in different stromal cell types.”

TNF-α produced by chemotherapy-activated stroma can amplify the CXCL1/2-S100A8/9 loop.

“Thus, TNF-α from chemotherapy-activated stroma can boost the CXCL1/2-S100A8/9 loop.”

Sca1 expression in CD11b+Ly6G+ populations indicates a potential progenitor-like phenotype of tumor granulocytic cells recruited by CXCL1/2.

“Sca1, a marker of the hematopoietic stem cells, was expressed in CD11b+Ly6G+ populations, indicating potential progenitor-like phenotype of the tumor granulocytic cells recruited by CXCL1/2 (Table S2).”

The NFκB/STAT1 pathway targets CXCL1/2 genes.

“CXCL1/2 are target genes of the NFκB/STAT1 pathway (Amiri and Richmond, 2003).”

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A recent Phase 2 breast cancer study found that non-responders to neoadjuvant chemotherapy and patients with residual disease had significantly higher circulating MDSC levels than responders.

“In line with our findings, recent Phase 2 study in breast cancer patients showed that non-responders of neoadjuvant chemotherapy and patients with residual disease had significantly higher circulating MDSC levels than did responders (Montero et al., 2011).”

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Such a paracrine survival network could benefit cancer cells under stress in both primary tumors and distant metastatic sites.

“Such a paracrine survival network could be beneficial to cancer cells under stress both in the primary tumor and at distant metastatic sites.”

The study identifies a new role for CD11b+Gr1+ cells in supporting metastatic breast cancer cell survival through S100A8/9 production.

“Our study delineates a new role for the CD11b+Gr1+ cells in mediating metastatic breast cancer cell survival through the production of S100A8/9.”

The paper's work links CXCL1/2 and S100A8/9 as functional partners in a paracrine loop between breast cancer cells and CD11b+Gr1+ myeloid cells that supports cancer cell survival during invasion or chemotherapy.

“In addressing this challenge, our work links CXCL1/2 and S100A8/9 as functional partners of a paracrine loop between breast cancer cells and CD11b+Gr1+ myeloid cells that supports the survival of cancer cells facing the rigors of invading new microenvironments or the impact of chemotherapy (Figure 7E).”

The present work illuminates how the tumor microenvironment responds to chemotherapy by hyperactivating a TNF-α-CXCL1/2-S100A8/9 paracrine axis that supports cancer cell survival under stress.

“The present work sheds light on how the tumor microenvironment responds to chemotherapy with hyperactivation of a TNF-α-CXCL1/2-S100A8/9 paracrine axis for cancer cell survival under stress.”

These results suggest that targeting the CXCL1/2-S100A8/9 axis may sensitize distant metastases to standard chemotherapy.

“Together, these results highlight the potential of targeting the CXCL1/2-S100A8/9 axis thereby sensitizing distant metastases to standard of care chemotherapy.”

• AssumptionRests on an assumption: CXCR2 is the primary receptor for CXCL1/2.

The findings functionally unify TNF-α, CXCL1/2, and S100A8/9 in a tumor-stroma paracrine axis that gives metastatic cells a survival advantage in stressed primary and metastatic microenvironments.

“Our findings functionally unify three important inflammatory modulators, TNF-α, CXCL1/2 and S100A8/9, in a tumor-stroma paracrine axis that provides a survival advantage to metastatic cells in stressed primary and metastatic microenvironments.”

The relevance of targeting the CXCL1/2-S100A8/9 axis may extend beyond breast cancer.

“Moreover, the important role of CXCR2 in pancreatic adenocarcinoma models (Ijichi et al., 2011) and of S100A8/9 in colorectal cancer (Ichikawa et al., 2011) suggest that the relevance of targeting the CXCL1/2–S100A8/9 axis may extend beyond breast cancer.”

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The authors' findings suggest that this cytokine burst includes TNF-α induced in several components of the tumor microenvironment after chemotherapy.

“Our current findings suggest that this cytokine burst includes TNF-α induced in several components of the tumor microenvironment after chemotherapy treatment.”

The paper's results provide mechanistic insights into the link between chemoresistance and metastasis in breast cancer.

“In conclusion, our results provide mechanistic insights into the link between two major hurdles in treating breast cancer: chemoresistance and metastasis.”

An adverse cycle involving TNF-α, CXCL1/2, and S100A8/9 could expand in response to chemotherapy.

“An adverse cycle involving TNF-α-CXCL1/2-S100A8/9 could thus be expanded in response to chemotherapy.”

These agents are a promising pharmacological approach in metastatic breast cancer when combined with standard chemotherapy.

“and we show that these agents are a promising pharmacological approach in metastatic breast cancer when combined with standard chemotherapeutic regimen.”

• AssumptionRests on an assumption: CXCR2 is the primary receptor for CXCL1/2.
• AssumptionRests on an assumption: Stroma has lower selective pressure for mutations and epigenetic changes than cancer cell genomes.

Stromal TNF-α has the undesirable effect of further increasing CXCL1/2 expression in breast cancer cells.

“An undesirable consequence of the stromal TNF-α is to further boost CXCL1/2 expression in breast cancer cells.”

Higher CXCL1/2 levels then drive the paracrine loop involving myeloid cell-derived S100A8/9 to enhance cancer cell survival.

“A higher level of CXCL1/2 then drives the paracrine loop involving myeloid cell-derived S100A8/9 to enhance cancer cell survival (Figure 7E).”

The effective combination of chemotherapy with CXCR2 inhibitors at metastatic sites in preclinical models suggests potential to limit disseminated tumor burden.

“The effective combination of chemotherapy with CXCR2 inhibitors at the metastatic site in our preclinical models underscores the potential application of this therapy to limit disseminated tumor burden.”

• AssumptionRests on an assumption: CXCR2 is the primary receptor for CXCL1/2.
• AssumptionRests on an assumption: Stroma has lower selective pressure for mutations and epigenetic changes than cancer cell genomes.