Lack of oxygen leads to more metastases

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Metastases are formed by cancer cells that break away from the primary tumor. A research group at the University of Basel has now identified lack of oxygen as the trigger for this process.

The results reveal an important relationship between the oxygen supply to tumors and the formation of metastases. This research may open up new treatment strategies for cancer.

The chances of recovery significantly worsen when a tumor metastasizes. Previous research has shown that metastases are formed by clusters of cancer cells that separate from the primary tumor and migrate to new tissue through the bloodstream.

However, thus far little has been known about why these clusters of circulating tumor cells (CTCs) leave the tumor in the first place.

Lack of oxygen leads to more metastases

Professor Nicola Aceto’s research group at the University of Basel’s Department of Biomedicine has now shown that a lack of oxygen is responsible for the separation of CTC clusters from the tumor.

This is an important starting point for the development of new cancer treatments.

A mouse model for breast cancer formed the basis of the experiments: the researchers analyzed the oxygen supply inside these tumors, which are equivalent to human cancer tissue, the detachment of CTCs and their molecular and cell biological properties.

It turned out that different areas of a tumor are supplied with different levels of oxygen: cancer cells with a lack of oxygen were found wherever the tumor had comparatively fewer blood vessels – in the core of the tumor as well as in clearly defined peripheral areas.

Next, the research team investigated the CTC clusters that had separated from these tumors and found that they similarly suffered from a lack of oxygen.

This led to the conclusion that cells leave the tumor if they do not receive enough oxygen.

“It’s as though too many people are crowded together in a small space. A few will go outside to find some fresh air,” says Aceto.

Further experiments showed that these CTC clusters with a lack of oxygen are particularly dangerous: in comparison to clusters with normal oxygen content, they formed metastases faster and shortened the mice’s survival time.

“If a tumor does not have enough oxygen, these CTC clusters, which have a particularly high potential to develop metastases, will break away,” says Aceto.

Stimulating blood vessel formation as a treatment approach

This insight led the researchers to take a closer look at the effect of what is called proangiogenic treatment: they stimulated the formation of blood vessels, thus boosting the supply of oxygen to the tumor cells.

As expected, the number of separating CTC clusters dropped, the mice formed fewer metastases, and they lived longer – but at the same time, the primary tumor increased in size significantly.

“This is a provocative result,” says Aceto. “If we give the tumor enough oxygen, the cancer cells have no reason to leave the tumor and metastasize. On the other hand, this accelerates the growth of the primary tumor.”

The next challenge is to transfer these findings to a clinical environment, where the characteristics of tumors vary from patient to patient: “But we speculate that substances that improve oxygen supply to the tumor can inhibit the formation of metastases in breast cancer, alone or in combination with other agents.”


More than half of solid tumors present with locally hypoxic or anoxic areas relative to the surrounding normal tissue [1]. Hypoxia has been associated with metastasis and poor prognosis in many cancers, including breast [1,2].

Tumor hypoxia results from an imbalance between oxygen delivered to the tumor niche and its consumption by cancer cells and tumor associated cells. Hypoxia develops in primary solid tumors due to multiple factors, including increased distance from blood supply, weakened vessel integrity, and competition for oxygen and nutrients from neighboring tumor and tumor-associated cells [1].

Both tumor and normal cells respond to a hypoxic environment by activating specific signaling pathways that lead to distinct gene expression changes, amongst the most immediate and salient common hubs being stabilization of the hypoxia-inducible factors, HIF-1α and HIF-2α [3].

In cancer, stabilized HIF-1α activates transcriptional programs that have been recognized to induce the epithelial to mesenchymal transition (EMT) and support metastasis in various cancer types [4–7]. Hypoxia-induced transcription factors and signaling pathways include Twist, Snail, ZEB1, Notch, TGF-β, and Hedgehog, among others[6,8–14].

Moreover, HIF1-α and hypoxia have been shown to increase the metastatic phenotype in multiple cancer cell types, including in breast cancer in vivo experiments, and have been linked to increased risk of metastasis and mortality in breast cancer patient cohorts [7,15–22].

Investigation of how the hypoxic tumor microenvironment contributes to increased cancer aggressiveness and metastatic potential may provide novel therapeutic avenues.

Hypoxia and subsequent stabilization of HIF-1α induce downstream metabolic changes in cancer cells. These include increased expression of glucose transporters and genes involved in glycolysis, altered fatty-acid and lipid metabolism, and increased pyruvate dehydrogenase kinase activity thereby decreasing the amount of pyruvate that enters the TCA cycle and decreasing oxidative phosphorylation [23–31].

One additional metabolic change is glycogen accumulation, which has been previously described in both cancerous and non-cancerous cells [32–35] under hypoxic conditions relative to their normal state. A variety of methods have been employed to exploit the potential vulnerabilities that arise from tumors’ adaptations to hypoxia, and have been shown to contribute to tumor control [36].

In this study, we focus on a deeper understanding of the potential vulnerabilities exhibited by the hypoxic modulation of glycogen homeostasis, carried out by a delicate balance between synthesizers and degraders of glycogen.

Glycogen is a high molecular weight branched polysaccharide of glucose and is the main glucose storage macromolecule in animals [37]. It is primarily stored in the liver where it is utilized to maintain blood-glucose levels and in the muscles where it can be mobilized quickly for energy production during exercise [37].

Glycogen is synthesized around a glycogenin core by addition of UDP-glucose onto growing glycogen chains. Glucose-1-phosphate available in the cell from either glucose transported into the cell or gluconeogenic substrates is catalyzed to UDP-glucose by UDP-glucose pyrophosphorylase-2 (UGP2). UDP-glucose is added onto glycogen via an α-1,4 linkage by glycogen synthase, the rate limiting enzyme in glycogen synthesis [37].

There are two isoforms of glycogen synthase: GYS1 which is mainly expressed in the liver and the muscle isoform GYS2. During degradation, glucose-1-phosphate molecules are removed from the non-reducing end of the glycogen molecule by glycogen phosphorylase 37, the rate limiting enzyme of glycogen degradation.

PYG has three different isoforms in humans that are typically expressed in different tissues: liver (PYGL), muscle (PYGM), and brain isoforms (PYGB). Free glucose-1-phosphate molecules are then catalyzed to glucose-6-phosphate, the first intermediate in glycolysis, by phosphoglucomutase-1 (PGM1).

To maintain glycogen-free glucose balance, glycogen synthase and glycogen phosphorylase are tightly regulated. Glycogen synthase activity is regulated allosterically and via posttranslational modification e.g. phsphorylation. Phosphorylation of glycogen synthase by glycogen synthase kinase 3α and 3β (GSK3α/β) at multiple serine residues inhibits its activity [38].

Glycogen phosphorylase is activated by phosphorylation at Ser-14 by phosphorylase kinase [37] and by the allosteric stimulator glucose-6-phosphate (G6P). Importantly, both glycogen synthase and glycogen phosphorylase are regulated in synchrony by protein phosphatase-1 (PP1).

PP1 dephosphorylation activates glycogen synthase, but inhibits glycogen phosphorylase, leading to reciprocal regulation of glycogen synthesis and degradation [37].

High levels of glycogen have been found in diverse cancer cell types including breast cancers [39]. Recently, glycogen levels were found to be inversely correlated with proliferation rate, indicating that glycogen was utilized as an energy source to sustain proliferation[39].

High levels of glycogen have also been found in the hypoxic tumor cores and in tumors treated with anti-angiogenic therapies [40]. Hypoxia and stabilization of HIF-1α have been shown to increase levels of many glycogen enzymes and regulatory proteins including UGP2, GYS1, GBE, and PPP1R3C, the glycogen-associated regulatory subunit 3C of PP1 [35,41–43].

Additionally, in work conducted by Favaro et al., siRNA knockdown of the liver isoform of glycogen phosphorylase was shown to inhibit glioblastoma cell proliferation under hypoxia and induce senescence in a reactive oxygen species-dependent manner [40]. While past studies have focused on glycogen accumulation as protection from the adverse hypoxic environment at the primary tumor site, our work aims to determine the relationship between glycogen accumulation and energy reserves utilized for hypoxia driven metastasis.

In this study, we sought to understand the link between the fuel provided by hypoxia-induced glycogen storage in aggressive breast cancers and the promotion of invasion and migration. We found that six different breast cancer cell lines and a normal-like breast epithelial cell line all increased their glycogen stores under hypoxia.

Glycogen gene expression changes under hypoxia were also evaluated, finding no consensus change in expression that would account for this increase, indicating other means of regulation of glycogen stores are in place, such as post-translational modification or allosteric regulation of the rate-limiting enzymes. In order to investigate how proliferation, migration, and invasion are affected by glycogen storage and utilization, we created glycogen phosphorylase knockdowns for both the liver and brain isoforms of PYG.

In the two breast cancer cell lines, MDA-MB-231 and MCF-7, loss of the brain isoform PYGB inhibited hypoxic glycogen usage whereas the loss of both PYGL and PYGB in the normal-like MCF-10A cell line exhibited this effect. Prohibition of glycogen utilization resulted in a marked decrease of proliferation in MCF-10A cells and a slight decrease in MCF-7 cells.

Wound-healing was strikingly decreased in shPYGB MCF-7 cells under both normoxic and hypoxic conditions. While loss of PYGB did not affect the proliferation or wound-healing of triple-negative breast cancer (TNBC) MDA-MB-231 cells, it did significantly decrease the invasiveness of these cells.

These findings indicate that attacking the cancer vulnerabilities derived from dysregulation of glycogen metabolism could be a therapeutic strategy, not only to slow tumor growth as has been previously suggested by other work, but also to inhibit development of distant metastases in breast cancers such as TNBC, for which few targeted therapies currently exist.

Discussion
It is well known that hypoxia increases migration, invasion, and metastasis in a variety of cancers, including breast cancers. Hypoxia also induces glycogen accumulation in cancer cells, promoting proliferation, protecting cells from reactive oxygen species, and preventing senescence [34,39,40].

Here we show that different types of breast cancer cells exhibit hypoxic glycogen accumulation and utilization of these glycogen stores contributes to proliferation, migration, and invasion.

All breast cancer cells tested increased glycogen stores in response to hypoxia. However, baseline normoxic glycogen levels and the amount of glycogen increase under hypoxic conditions varied widely between cell lines, with no discernible pattern based on receptor status or sub-type.

Inflammatory breast cancer cells increased their glycogen stores in response to hypoxia by over 10-fold higher than other breast cancer cell types, suggesting that interventions based on inhibiting glycogen utilization may be more damaging to this aggressive breast cancer subtype.

Overall, these data indicate that glycogen metabolism phenotypes in breast cancer may vary widely depending on each individual tumor. Even though there is no pattern to glycogen metabolism that is distinct for the commonly used breast cancer biomarkers, relative glycogen levels in patient biopsies can be determined by a simple and reliable histological test (PAS staining), thus potentially facilitating patient selection for interventions based on glycogen metabolism in the future.

We also found no single consensus glycogen gene expression signature that would account for the hypoxic glycogen accumulation observed in our breast cancer cells, indicating that there will be high degree of heterogeneity in the regulation of the common event we describe of glycogen storage under hypoxia.

Previous studies have proposed that glycogen accumulation in breast cancer cells is due to HIF1α mediated increase in GYS1 and/or PPP1R3C expression in hypoxia [42,43]. In agreement with those prior results, we found an increase in GYS1 expression in MCF-7 cells and increased PPP1R3C expression in SUM-149 and normal-like MCF-10A cells; however, importantly, we find that there is no consensus glycogen-related gene expression among all breast cancer cells that leads to the observed hypoxic increase in glycogen.

This result is important because it suggests that modulation of the rate limiting reactions of glycogen synthesis or degradation, rather than interventions on upstream targets, would have more general utility in breast cancer. This accumulation of glycogen could also be caused by allosteric regulation or phosphorylation/dephosphorylation of the rate-limiting enzymes of glycogen metabolism.

Future studies will need to determine the exact mechanism of hypoxic glycogen accumulation based on glycogen synthase and glycogen phosphorylase regulation and the possible relation to HIF1α stabilization under hypoxia in breast cancer, in a context dependent manner.

Regardless of the mechanism of hypoxic glycogen accumulation, we successfully inhibited glycogen utilization in breast cancer using shRNA knockdown of the glycogen phosphorylase isoforms PYGL and PYGB. Previous work in the field focused solely on the liver isoform of glycogen phosphorylase [40].

However, we determined that the brain isoform of glycogen phosphorylase is primarily responsible for glycogen degradation in MDA-MB-231 and MCF-7 breast cancer cells and both isoforms contribute in normal-like MCF-10A cells. This novel finding should inform future glycogen metabolism studies in breast and other cancers to include all isoforms of glycogen phosphorylase in addition to the well-studied liver isoform.

Inhibition of glycogen utilization also led to drastic phenotypic changes in breast cancer cells in both hypoxic and normoxic conditions. Proliferation was reduced in shPYGB MCF-7 cells and both shPYGL and shPYGB normal-like MCF-10A cells, which matches with the inhibition of glycogen utilization seen in these cells.

In the ER+ MCF-7 breast cancer cell line, wound-healing was also inhibited in the shPYGB cells. Wound-healing assays measure the ability of cells to move and grow outwards from an area of dense cell population. Without the ability to utilize glycogen, MCF-7 cells were unable to close the wound as efficiently as the control or the shPYGL cells.

This effect was not seen in the non-cancerous, normal-like breast epithelial MCF-10A cells, indicating that glycogen usage to promote migration is a cancer-specific phenotype and thus a possible vulnerability.

Additionally, in the TNBC MDA-MB-231 cells, inhibition of glycogen utilization by PYGB knockdown led to a significant decrease in invasive potential, reaffirming the importance of the brain isoform, PYGB, in advantaging cancer cells to more aggressive phenotypes based on enhanced glycogen availability.

While all breast cells tested increased glycogen storage under hypoxia, glycogen utilization as promoted by the brain isoform of glycogen phosphorylase, PYGB, affects migration and invasion phenotypes only in cancer cells and not in normal-like epithelial cells. These findings suggest PYGB as a potential novel target to reduce invasiveness and metastasis of breast cancers.

Future work will focus on recapitulating these in vitro results in in vivo tumor xenograft and metastasis studies, as well as investigating the anti-metastatic effects of treatments with glycogen phosphorylases inhibitors, such as ingliforib [46].

reference link : https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0220973


More information: Cell Reports (2020). DOI: 10.1016/j.celrep.2020.108105

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