Molecular profiling of a lethal tumor microenvironment, as defined by stromal caveolin-1 status in breast cancers

Abstract

Breast cancer progression and metastasis are driven by complex and reciprocal interactions, between epithelial cancer cells and their surrounding stromal microenvironment. We have previously shown that a loss of stromal Cav-1 expression is associated with an increased risk of early tumor recurrence, metastasis and decreased overall survival. To identify and characterize the signaling pathways that are activated in Cav-1 negative tumor stroma, we performed gene expression profiling using laser microdissected breast cancer-associated stroma. Tumor stroma was laser capture microdissected from 4 cases showing high stromal Cav-1 expression and 7 cases with loss of stromal Cav-1. Briefly, we identified 238 gene transcripts that were upregulated and 232 gene transcripts that were downregulated in the stroma of tumors showing a loss of Cav-1 expression (p ≤ 0.01 and fold-change ≥ 1.5). Gene set enrichment analysis (GSEA) revealed "stemness," inflammation, DNA damage, aging, oxidative stress, hypoxia, autophagy and mitochondrial dysfunction in the tumor stroma of patients lacking stromal Cav-1. Our findings are consistent with the recently proposed "Reverse Warburg Effect" and the "Autophagic Tumor Stroma Model of Cancer Metabolism." In these two complementary models, cancer cells induce oxidative stress in adjacent stromal cells, which then forces these stromal fibroblasts to undergo autophagy/mitophagy and aerobic glycolysis. This, in turn, produces recycled nutrients (lactate, ketones and glutamine) to feed anabolic cancer cells, which are undergoing oxidative mitochondrial metabolism. Our results are also consistent with previous biomarker studies showing that the increased expression of known autophagy markers (such as ATG16L and the cathepsins) in the tumor stroma is specifically associated with metastatic tumor progression and/or poor clinical outcome.

Figure 1

Stromal Cav-1 can be used to stratify human breast cancer patients into two transcriptionally distinct patient populations. The transcriptional profiles of Cav-1-positive (+) tumor stroma (N = 4) versus Cav-1-negative (−) tumor stroma (N = 7) were compared. We identified 238 gene transcripts that were upregulated and 232 gene transcripts that were downregulated in the stroma of tumors showing a loss of Cav-1 expression (Sup. Table 1). Note that the two patient populations are transcriptionally different. One-way ANOVA was setup to extract differentially expressed genes between Cav-1 positive and Cav-1 negative samples. The resultant p-values were further adjusted by multi-test correction (MTC) method of FDR step-up. The standardized intensity data from the stringent gene list (p-value ≤ 0.01 and fold change ≥1.5) were used in generating the hierarchical clustering HeatMap.

Figure 2

HeatMaps of gene transcripts associated with myofibroblast differentiation, autophagy, lysosomal degradation and glycolysis. Note that Cav-1-deficient stroma shows the upregulation of myofibroblast differentiation (15 transcripts), autophagy (22 transcripts), lysosomal proteases (5 transcripts), lysosomal proteins (29 transcripts) and glycolysis/pyruvate metabolism (15 transcripts). See Supplemental Tables 4, 5 and 10.

Figure 3

HeatMaps of gene transcripts associated with the response to hypoxia and mitochondria. Note that Cav-1-deficient stroma shows the upregulation of hypoxia target genes (65 transcripts) and mitochondrial-associated proteins (41 transcripts). See Supplemental Tables 10 and 12.

Figure 4

HeatMaps of gene transcripts associated with inflammation and redox/stress signaling. Note that Cav-1-deficient stroma shows the upregulation of TNF/NFκB signaling (11 transcripts), the immune response (31 transcripts) and redox/stress signaling (19 transcripts). See Supplemental Tables 6 and 10.

Figure 5

HeatMaps of gene transcripts associated with DNA damage and repair. Note that Cav-1-deficient stroma shows the upregulation of the DNA damage response (67 transcripts). See Supplemental Table 11.

Figure 6

HeatMaps of gene transcripts associated with aging, apoptosis and BRCA1 mutation-positive breast cancer patients. Note that Cav-1-deficient stroma shows the upregulation of aging (73 transcripts), apoptosis (51 transcripts) and BRCA1-mutation associated genes (20 transcripts). See Supplemental Tables 7, 9 and 14.

Figure 7

HeatMaps of gene transcripts associated with ER-negative breast cancers. Note that Cav-1-deficient stroma shows the upregulation of genes associated with ER-negative breast cancers (96 transcripts). See Supplemental Table 8.

Figure 8

HeatMaps of gene transcripts associated with neural stem cells. Note that Cav-1-deficient stroma shows the upregulation of genes normally associated with neural stem cells (202 transcripts). See Supplemental Table 13.

Figure 9

Venn diagrams for the intersection of the Cav-1-deficient stromal gene signature with other breast cancer tumor stromal gene sets. We compared the gene transcripts upregulated in Cav-1-deficient tumor stroma (1,819 transcripts encoding 1,297 unique genes; p ≤ 0.05 and fold-change (f.c.) ≥ 1.15; Sup. Table 3) with (upper) tumor stromal and (lower) recurrence stromal gene lists, as defined in the text of the manuscript. Note that the gene transcripts upregulated in Cav-1-deficient tumor stroma show significant overlap with tumor stroma (a 440 transcript overlap; p = 4.76 × 10⁻⁹) and recurrence stroma (a 214 transcript overlap; p < 0.001).

Figure 10

The Cav-1-deficient stromal gene signature is upregulated in breast cancer and is associated with tumor recurrence. In (A and B), box-plots show that the Cav-1-deficient stromal signature is upregulated in all breast cancers, both ER(+) and ER(−) sub-types, relative to normal healthy breast tissue. In (A), ER status was determined by immuno-histochemistry, while in (B), ER status was inferred from ESR1 transcript expression. In (C), the Cav-1-deficient stromal signature was associated with increased recurrence in breast cancer patients. In (A–C), we used the Cav-1-deficient stromal signature included in Supplemental Table 1 (238 transcripts that were specifically upregulated; p ≤ 0.01 and fold-change (f.c.) ≥ 1.5). Qualitatively similar results were also obtained with the longer signature included in Supplemental Table 3. IQR, inter-quartile

Figure 11

The Cav-1-deficient stromal gene signature is associated with tumor recurrence and poor survival in ER(+) and luminal A breast cancer patients. Note that the Cav-1-deficient stromal signature (238 transcripts that were specifically upregulated; p ≤ 0.01 and fold-change (f.c.) ≥ 1.5; Sup. Table 1) is clearly associated with increased recurrence (A and C) and decreased overall survival (B and D), despite the fact that these breast cancer-derived tumors were not subjected to laser capture microdissection. (A and B) are ER(+) breast cancer patients, while (C and D) are the luminal A subset of ER(+) breast cancer patients. Qualitatively similar results were also obtained with the longer signature included in Supplemental Table 3.

Figure 12

Understanding the hierarchy of the cellular processes associated with a Cav-1-deficient tumor microenvironment. Oxidative stress in fibroblasts is known to be sufficient to induce (1) myo-fibroblast differentiation, (2) DNA damage and (3) a pseudo-hypoxic state.,–,, This pseudohypoxic state in fibroblasts then leads to the activation of NFκB and HIF-1α, which are master regulators of the immune response and mitochondrial function, as well as autophagy.– The autophagic destruction of mitochnodria then drives aerobic glycolysis. We have previously shown that transient knock-down of Cav-1 in fibroblasts, using a targeted siRNA-approach, is sufficient to induce myo-fibroblast differentation, DNA damage and ROS production, leading to a pseudo-hypoxic state.– Similarly, knock down of Cav-1 in fibroblasts is sufficient to drive NFκB- and HIF1-activation, as well as mitochondrial dys-function, autophagy and the induction of glycolytic enzymes.– Interestingly, transcriptional profiling of a Cav-1-deficent tumor microenvironment provides direct evidence to support the involvement of all of these biological processes (See HeatMaps in Figs. 2–6). The red arrow denotes that NFκB-activation is known to augment HIF1-activation and visa versa, indicating that they act synergistically.