Understanding the "lethal" drivers of tumor-stroma co-evolution

Abstract

We have recently proposed a new model for understanding how tumors evolve. To achieve successful "Tumor-Stroma Co-Evolution", cancer cells induce oxidative stress in adjacent fibroblasts and possibly other stromal cells. Oxidative stress in the tumor stroma mimics the effects of hypoxia, under aerobic conditions, resulting in an excess production of reactive oxygen species (ROS). Excess stromal production of ROS drives the onset of an anti-oxidant defense in adjacent cancer cells, protecting them from apoptosis. Moreover, excess stromal ROS production has a "Bystander-Effect", leading to DNA damage and aneuploidy in adjacent cancer cells, both hallmarks of genomic instability. Finally, ROS-driven oxidative stress induces autophagy and mitophagy in the tumor micro-environment, leading to the stromal over-production of recycled nutrients (including energy-rich metabolites, such as ketones and L-lactate). These recycled nutrients or chemical building blocks then help drive mitochondrial biogenesis in cancer cells, thereby promoting the anabolic growth of cancer cells (via an energy imbalance). We also show that ketones and lactate help "fuel" tumor growth and cancer cell metastasis and can act as chemo-attractants for cancer cells. We have termed this new paradigm for accelerating tumor-stroma co-evolution, "The Autophagic Tumor Stroma Model of Cancer Cell Metabolism". Heterotypic signaling in cancer-associated fibroblasts activates the transcription factors HIF1alpha and NFκB, potentiating the onset of hypoxic and inflammatory response(s), which further upregulates the autophagic program in the stromal compartment. Via stromal autophagy, this hypoxic/inflammatory response may provide a new escape mechanism for cancer cells during anti-angiogenic therapy, further exacerbating tumor recurrence and metastasis.

Biomarker Studies: Identification of a “Lethal Tumor Microenvironment”

We recently identified that a loss of stromal caveolin-1 (Cav-1) is a powerful independent biomarker for predicting clinical outcome in human breast cancer patients.1–4 More specifically, a loss of stromal Cav-1 effectively predicts early tumor recurrence, lymph-node (LN) metastasis, lympho-vascular invasion (LVI) and tamoxifen-resistance and, as a consequence, is associated with poor clinical outcome in human breast cancer patients.1 Importantly, the prognostic value of a loss of stromal Cav-1 is independent of epithelial marker status (ER, PR, HER2), making it a valuable biomarker in all the most common sub-types of breast cancer,1 including triple negative (TN) and basal-like breast cancer.2 For example, >75% of TN patients with high stromal Cav-1 have an overall survival of nearly 12 years.2 Conversely, <10% of TN patients with absent stromal Cav-1 remain alive 5 years post-diagnosis.2 Thus, a loss of stromal Cav-1 in human breast cancers is associated with a “lethal stromal phenotype”. Similar results were obtained with DCIS (a breast cancer precursor lesion),4 and prostate cancer,5 providing an indication that the predictive value of stromal Cav-1 may extend to a wide variety of other tumor types.

The “Autophagic Tumor Stroma Model of Cancer”: Co-Culture Studies with Fibroblasts and Mouse Animal Models

To understand the molecular basis of the lethality of a loss of stromal Cav-1, we developed a co-culture system to mimic tumor-stroma co-evolution.6 In this co-culture system, stromal fibroblasts (hTERT-BJ1 cells) were mixed with human breast cancer cells (MCF-7) and allowed to interact for 3–5 days.6 Using this simplified model system, we observed that cancer cells induce oxidative stress in adjacent stromal fibroblasts, with the over-production of reactive oxygen species (ROS) and local DNA damage7,8 (Fig. 1).

Oxidative stress in stromal fibroblasts, in turn, induced an anti-oxidant defense in neighboring cancer cells, dramatically protecting them against apoptosis7,8 (Fig. 1). In this regard, we observed that the fibroblasts specifically induce anti-oxidant (peroxiredoxin1) and anti-apoptotic proteins (TIGAR) in adjacent cancer cells.7,8 However, ROS over-production in cancer-associated fibroblasts also had a “Bystander Effect”, driving DNA damage (gamma-H2AX staining; a marker of DNA double-strand breaks) and aneuploidy in cancer cells.7,8 These are hallmarks of genomic instability. Thus, fibroblasts protect cancer cells against apoptosis and at the same time, allow cancer cells to undergo random mutagenesis, to accelerate tumor-stroma co-evolution7,8 (Fig. 1). In accordance with this hypothesis, genomic alterations are frequently observed both in tumor cells and the surrounding stromal cells.9,10

Finally, we also observed that cancer cells induce a loss of Cav-1 and mitochondria in adjacent fibroblasts.7,8 This appears to be due to the onset of autophagy, as both anti-oxidants (such as N-acetyl-cysteine (NAC), metformin and quercetin), as well as lysosomal/autophagy inhibitors (chloroquine) can prevent this process.7,8 Autophagy in cancer associated fibroblasts provides adjacent tumor cells with energy-rich recycled nutrients and chemical building blocks to fuel the anabolic growth of cancer cells.7,8 Since these cancer-associated fibroblasts lose their mitochodria via mitophagy (the autophagic destruction of mitochondria), they are forced to undergo aerobic glycolysis, resulting in the stromal production of pyruvate, lactate and ketone bodies (3-hydroxybutyrate).7,8,11 To describe this phenomenon, we have coined the term, “The Reverse Warburg Effect,”12–16 as the conventional Warburg Effect was thought to be confined to cancer cells and not to occur in stromal fibroblasts. Importantly, these energyrich metabolites can then be transferred to adjacent cancer cells, where they fuel oxidative mitochondrial metabolism, the TCA cycle and ATP production, as well as additional mitochondrial biogenesis.12–16 This results in a unilateral, vectorial, net energy transfer from the catabolic tumor stroma to anabolic cancer cells. Thus, autophagy in the tumor stroma results in a negative energy balance, in favor of the cancer cells.11 We have termed this new mechanism underlying tumor-stroma co-evolution, “The Autophagic Tumor Stroma Model of Cancer Cell Metabolism”11(Fig. 1).

Using a variety of molecular and genetic approaches, we showed that oxidative stress drives the induction of HIF1alpha and NFκB-activation in cancer associated fibroblasts, leading to the onset of autophagy and mitophagy.7,8,11,15 For example, we see that cancer cells activate HIF1- and NFκB-responsive luciferase reporters in adjacent stromal fibroblasts.8 Moreover, genetic activation of HIF1alpha or NFκB in stromal cells was sufficient to confer the cancer-associated fibroblasts phenotype, resulting in 2–3-fold increases in epithelial tumor growth,17 as well as the onset of local lymph node metastasis, without any detectable increases in tumor angiogenesis.17 Finally, direct administration of energy-rich metabolites (such as L-lactate and 3-hydroxy-butyrate) was sufficient to increase tumor volume and metastasis,18 without any increases in tumor angiogenesis.18

Importantly, we validated that acute loss of Cav-1, using an siRNA approach, is indeed sufficient to induce ROS production and mitochondrial dysfunction in human fibroblasts7 (Fig. 2). Similarly, acute loss of Cav-1, using the same approach, is sufficient to activate autophagy and mitophagy8 (Fig. 3A). Under these conditions, we see the upregulation of both autophagy (Beclin1) and mitophagy (BNIP3 and BNIP3L) markers8 (Fig. 3A). Thus, oxidative stress and autophagy acutely downregulate Cav-1 by autophagic/lysosomal degradation6 and a loss of Cav-1 further exacerbates these stressors, driving more oxidative stress, mitochondrial dysfunction and autophagy—in a feed-forward fashion.7,8 Consistent with these findings, human breast cancers that lack stromal Cav-1 show the stromal overexpression of markers for (1) aerobic glycolysis (PKM2/LDH-B)12,18 and (2) mitophagy (BNIP3L)8 (Fig. 3B). The selective stromal expression of PKM2 and LDH-B was also validated using a xenograft model employing GFP-tagged MDA-MB-231 cells,7,17 which are an aggressive metastatic triple-negative/basal breast cancer cell line.

One mechanism by which an acute loss of Cav-1 generates oxidative stress is via increased NO (nitric oxide) production,7,15 as Cav-1 is a natural endogenous inhibitor of NOS (nitric oxide synthase).19 Increased NO production, in turn, generates ROS and oxidative stress via mitochondrial dysfunction, by inhibiting mitochondrial oxidative phosphorylation via Complex I and Complex IV. Interestingly, we have previously shown that a loss of Cav-1 induces the tyrosine nitration of mitochondrial Complex I,15 consistent with our hypothesis. Furthermore, metabolic restriction with glycolysis (2-DG) and Complex I (metformin) inhibitors is synthetically lethal with a Cav-1 deficiency in mice.15 Thus, Cav-1 (-/-) null mice have a severely reduced mitochondrial reserve capacity.15

As predicted based on the above studies, mammary fat pads derived from Cav-1 (-/-) null mice show the upregulation of markers for hypoxia, oxidative stress and autophagy.8,11 Metabolomic analysis of the Cav-1 (-/-) mammary fat pad demonstrates the upregulation of nearly 100 metabolites, consistent with a major catabolic phenotype.11 Their metabolic profile is consistent with constitutive oxidative stress and mitochondrial dysfunction, with the elevation of markers of oxidative stress (ADMA) and mitochondrial dysfunction (3-hydroxy-butyrate).11 Thus, ADMA and ketone production are features of a lethal tumor microenvironment.11 This may explain why patients with diabetes, that show increased levels of ADMA and ketones, as well as oxidative stress and autophagy, have an increased risk for the development of a variety of epithelial cancer sub-types.11

Lastly, an informatics analysis of the unbiased transcriptional profiles of tumor stroma isolated from human breast cancers shows the overexpression of markers of oxidative stress, hypoxia and autophagy, as well as lysosomal enzymes.11,16 This provides further translational validation of the “Autophagic Tumor Stroma Model of Cancer.”11 Many of these markers are also associated with breast cancer tumor recurrence and metastasis. In fact, the transcriptional profile of autophagic Cav-1 (-/-) null mesenchymal stem cells is most closely related to (1) the primary tumor stroma derived from breast cancer patients that have undergone metastasis; and (2) the brains of patients with Alzheimer disease.16 Consistent with these findings, the pathogenesis of Alzheimer disease is thought to involve ROS and NO over-production (oxidative stress) and mitochondrial dysfunction, as well as autophagy.16 In fact, the Alzheimer disease gene list is also most closely related to the tumor stroma isolated from patients with metastatic breast cancer,16 as compared to patients without metastatic disease.

Implications for Cachexia and Effective Cancer Chemotherapy

The “Autophagic Tumor Stroma Model of Cancer” also has important implications for understanding cancer-associated cachexia and improving cancer chemotherapy. Cancerassociated cachexia, also known as systemic wasting, occurs in patients with advanced and metastatic cancer.20–24 Cachexia is due to increased energy expenditures and an increased metabolic rate, which results in a negative energy balance.20–24 Thus, we believe that autophagy in the tumor stroma represents the local microscopic equivalent of cancer-associated cachexia and may explain how cachexia could start locally and then spread systemically. In accordance with this hypothesis, others have previously suggested that cancer-associated cachexia is caused by oxidative stress and is responsive to treatment with anti-oxidants.25–35

Regarding cancer chemotherapy, we propose that anti-oxidants and autophagy/lysosomal inhibitors could be used systemically to halt autophagy in the tumor stroma, effectively cutting off the tumor's fuel supply. In essence, anti-oxidants and autophagy inhibitors would metabolically uncouple the autophagic tumor stroma from adjacent anabolic “parasitic” cancer cells. Thus, new clinical trials with anti-oxidants (such as N-acetyl-cysteine, metformin and quercetin), as well as lysosomal inhibitors (chloroquine) may be warranted. Importantly, these drugs are either available over-the-counter (OTC) as dietary supplements or are FDA-approved drugs.

Understanding Why Anti-Angiogenic Drugs Paradoxically Promote Tumor Progression and Metastasis

Finally, our current model of tumor-stroma co-evolution may also explain the clinical failure of anti-angiogenic drugs, such as Bevacizumab (Avastin). Effective anti-angiogenic therapies induce hypoxia in the tumor stroma.36–49 Hypoxia in the tumor stroma then induces autophagy. We have shown that hypoxia and autophagy in the tumor stromal microenvironment are the necessary “ingredients” for driving the development of a “lethal tumor stromal phenotype.” Thus, autophagy in the tumor stroma provides a new escape mechanism for cancer cells during anti-angiogenic therapy,14 which could then further drive more aggressive tumor progression and metastasis.14 This explains how anti-angiogenic therapy could convert a non-aggressive tumor into a lethally aggressive metastatic cancer, by fueling tumor-stroma co-evolution.

Figures and Tables

Figure 1 Deciphering tumor-stroma co-evolution: the autophagic tumor stroma model of cancer cell metabolism. Cancer cells induce oxidative stress in adjacent stromal fibroblasts. Then, the resulting oxidative stress in cancer-associated fibroblasts helps drive tumor-stroma co-evolution, by randomly mutagenizing cancer cells, while protecting them against apoptosis and providing them with abundant recycled nutrients and chemical building blocks via autophagy. This results in a net energy transfer from the autophagic tumor stroma to the anabolic “hungry” cancer cells. Thus, stromal oxidative stress and autophagy function(s) as a “battery” to drive tumor-stroma co-evolution and “fuel” oxidative mitochondrial metabolism in cancer cells. A+ (positive), increased autophagy/mitophagy in cancer associated fibroblasts; A− (negative), decreased autophagy/mitophagy in epithelial cancer cells. AR (resistant), denotes the successful evolution of autophagy-resistant cancer cells, due to genetic silencing or deletion of required autophagy genes, such as Beclin1.

Figure 2 Acute knock-down of Cav-1 in human stromal fibroblasts increases ros production and negatively affects mitochondrial activity. (Upper) Cav-1 knock-down induces ROS production. CM-H₂DCFDA staining (green) was performed on hTER T-fibroblasts treated with Cav-1 siRNA (right) or control siRNA (left). Cells were counterstained with Hoechst nuclear stain (blue). Samples were then immediately imaged using a 488 nm excitation wavelength. Note that Cav-1 knock-down greatly promotes ROS generation. Importantly, images were acquired using identical exposure settings. Original magnification, 20x. (Lower) Cav-1 knock-down decreases mitochondrial activity. hTERT-fibroblasts were treated with Cav-1 siRNA (right) or control siRNA (left). Then, functional mitochondria with active membrane potential were visualized using MitoTracker staining (red). DAPI was used to stain nuclei (blue). Note that transient Cav-1 knock-down greatly decreases mitochondrial activity. Importantly, paired images were acquired using identical exposure settings. Original magnification, 63x. Images were reproduced from references,7 and 7 with permission.

Figure 3 Acute knock-down of Cav-1 in human stromal fibroblasts activates autophagy and mitophagy: implications for human breast cancer. (A) Acute loss of Cav-1 increases the expression of autophagic markers. hTER T-fibroblasts were treated with Cav-1 siRNA or control (CTR) siRNA. Cells were fixed and immuno-stained with antibodies against Beclin1, BNIP3 and BNIP3L. DAPI was used to visualize nuclei (blue). Importantly, paired images were acquired using identical exposure settings. Original magnification, 40x. Note that acute Cav-1 knockdown is sufficient to greatly increase the expression levels of all the autophagy/mitophagy markers we examined. (B) BNIP3L is highly increased in the stroma of human breast cancers that lack stromal Cav-1. Paraffin-embedded sections of human breast cancer samples lacking stromal Cav-1 were immuno-stained with antibodies directed against BNIP3L. Slides were then counter-stained with hematoxylin. Note that BNIP3L is highly expressed in the stromal compartment of human breast cancers that lack stromal Cav-1. Original magnification, 20x and 40x, as indicated. Images were reproduced from the references 7 and 8, with permission.

Acknowledgements

F.S. and her laboratory were supported by grants from the W.W. Smith Charitable Trust, the Breast Cancer Alliance (BCA) and a Research Scholar Grant from the American Cancer Society (ACS). M.P.L. was supported by grants from the NIH/NCI (R01-CA-080250; R01-CA-098779; R01-CA-120876; R01-AR-055660) and the Susan G. Komen Breast Cancer Foundation. A.K.W. was supported by a Young Investigator Award from Breast Cancer Alliance, Inc., and a Susan G. Komen Career Catalyst Grant. R.G.P. was supported by grants from the NIH/NCI (R01-CA-70896, R01-CA-75503, R01-CA-86072 and R01-CA-107382) and the Dr. Ralph and Marian C. Falk Medical Research Trust. The Kimmel Cancer Center was supported by the NIH/NCI Cancer Center Core grant P30-CA-56036 (to R.G.P.). Funds were also contributed by the Margaret Q. Landenberger Research Foundation (to M.P.L.). This project is funded, in part, under a grant with the Pennsylvania Department of Health (to M.P.L. and F.S.). The Department specifically disclaims responsibility for any analyses, interpretations or conclusions. This work was also supported, in part, by a Centre grant in Manchester from Breakthrough Breast Cancer in the UK (to A.H.) and an Advanced ERC Grant from the European Research Council.