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Skeletons Come out of the Closet to Fight Cancer

Cancer cells divide rapidly and uncontrollably.  Anticancer drugs that target the microtubule cytoskeleton work by preventing cancer cells from dividing correctly, but they also affect other rapidly dividing healthy cells and some aggressive cancers are resistant to their effects. New insights are revealing how specific features of the microtubule cytoskeleton are making cancer cells more aggressive and difficult to treat, paving the way for new anticancer therapies. Credit: Mopic/adobe

Cancer cells divide rapidly and uncontrollably. Anticancer drugs that target the microtubule cytoskeleton work by preventing cancer cells from dividing correctly, but they also affect other rapidly dividing healthy cells and some aggressive cancers are resistant to their effects. New insights are revealing how specific features of the microtubule cytoskeleton are making cancer cells more aggressive and difficult to treat, paving the way for new anticancer therapies. Credit: Mopic/adobe

By Amelia Parker

Cells have skeletons that hold their shape and help them move around. Recent discoveries have revealed that a protein in some cytoskeletons is making cancer cells more deadly, fundamentally challenging our understanding of the function of the cell’s skeleton and offering new hope for the development of targeted and effective cancer therapies.

Despite decades of advances in the way we understand, diagnose and treat cancer, a cancer diagnosis is one of uncertainty. Not all cancers behave in the same way or respond in the same way to treatment, leaving patients without assurances that the treatments that they endure will work, all while suffering through debilitating and potentially lifelong side-effects. Improvements in cancer treatments will depend on our ability to accurately predict which treatments will work most effectively in which patients.

Far from being a benign structural entity, the cell skeleton, or cytoskeleton, has now emerged as an important determinant of cancer’s aggression, making it a target for the next generation of specific anticancer treatments.

No Bones About It, Cells Have Skeletons Too

Unlike the bones that make up our body’s skeleton, the cytoskeleton is made up of a mesh of protein filaments that span the crowded interior of the cell. These filaments are composed of different types of proteins arranged like beads on a string. These form an elaborate interconnected network that holds the shape of the cell and helps cells move around.

Microtubules are one of the main types of filaments that form this cytoskeletal network. Although these microtubule fibres play a fundamental role in the processes that keep cells alive and functioning, they are also emerging as an important player in the processes that make cancers more aggressive and resistant to treatment.

In healthy cells that aren’t dividing, microtubule fibres act as tracks for the cell’s motors to transport cargo from one side of the cell to the other, ensuring that each region of the cell can obtain the nutrients and energy required. In cells that are dividing, the microtubule cytoskeleton ensures that the subsequent generation of cells contain the full complement of the genes necessary for a cell to function.

During cell division, the microtubules physically attach to the chromosomes and pull each chromosome set to opposite sides of the cell, so that when this parent cell splits down the middle to form two daughter cells, each daughter cell receives one complete set of chromosomes. To achieve this, microtubule filaments must be very dynamic, lengthening and shortening to drive the chromosomes to each side of the cell.

It is this function that brought microtubules to the centre of attention for the development of anticancer drugs in the 1950s and 1960s. Drugs that target the microtubule cytoskeleton have since become the most widely used chemotherapies in the world, and are still used extensively as the first-line therapy for many common cancers, including breast, ovarian and lung cancer. These drugs kill rapidly dividing cancer cells by binding directly to microtubules, restraining their dynamic behaviour and preventing them from separating chromosomes during cell division. This causes cells to stop dividing and die.

However, since these drugs bind indiscriminately to all microtubules, they therefore target the cytoskeletons of all cells. Their effects on rapidly dividing healthy cells, such as cells that line the gastrointestinal tract or form hair follicles, result in severe and debilitating side-effects that can limit their use in patients.

Cancers can also be resistant to these drugs, further limiting their effectiveness. The reasons why some cancers are resistant to these drugs are not clear but appear to be related to the types of proteins that make up the microtubule cytoskeleton. By identifying how the cancer cell cytoskeleton differs from those of healthy cells in finer detail, we have the opportunity to develop more specific drugs and targeted therapeutic strategies that will more effectively treat a wide range of common cancers while minimising the side-effects associated with these treatments.

Revealing the Finer Points of Difference

Each microtubule filament is composed of different types of tubulin proteins. There are eight different types of α-tubulin and seven different types of β- tubulin proteins in humans. These different tubulins can be thought of as differently coloured beads strung together to form a microtubule filament.

Different types of cells contain different mixtures of tubulin proteins. For example, along with a mixture of other β-tubulin proteins, nerve cells contain the βIII-tubulin protein, which is not found in other cell types.

It’s believed that the types of tubulin proteins that form microtubule filaments within cells constitute instructions to control the structure and dynamics of the microtubule network. However, understanding what these instructions mean and how cells interpret them has remained elusive for the past several decades. Now precise measurements revealing changes in the types of tubulin proteins in cancer cells compared with normal healthy cells is shedding new light on the function of different tubulin proteins, and exposing opportunities for the development of new cancer treatments.

The composition of the cytoskeleton is different in cancer cells and healthy cells. Cancer cells express different types of tubulin proteins compared with normal cells in the same organs. In particular, many different types of cancer cells express the βIII-tubulin protein, which is not normally found in normal healthy cells of the same organs. Importantly, high levels of the βIII-tubulin protein make cancers more aggressive and more difficult to treat.

Since the first report nearly 20 years ago that βIII-tubulin is expressed in ovarian cancer cells that are resistant to standard treatment, the association between this protein and aggressive, treatment-resistant cancers has been extended to include many of the deadliest cancers, including lung and breast cancers. As a type of tubulin that is normally only expressed in nerve cells and in specialised cells of the testes, these observations have baffled scientists and doctors alike. With few clues as to what this protein normally does in healthy nerve cells, understanding what this protein is doing to make cancer cells more aggressive and resistant to treatment has remained a major barrier to identifying treatments for cancers with high levels of this protein.

Several key studies in cancer cells and mouse models of lung, pancreatic and breast cancers over recent years have recapitulated this clinical data, showing that βIII-tubulin protects cells from a broad range of chemotherapy agents, promotes tumour formation and increases the metastatic potential of cancer cells. These studies indicated that βIII-tubulin is conferring a survival advantage to cancer cells, providing compelling evidence that the function of βIII-tubulin extends beyond its structural role in cells. However, the studies could not explain how βIII-tubulin exert these effects, a conundrum that remained puzzling.

Cancer Cells Are Not So Sweet

A key piece of the puzzle proved to be considering how cancer cells respond to the environment of the tumour. Tumour tissue is not like the surrounding healthy tissue. Cancer cells grow much faster than healthy cells, and their rapid proliferation make them ravenous for more nutrients such as glucose. To support this growth they instruct the blood vessels to branch out and grow further, supplying them with more blood containing the nutrients they require. However, the blood vessels are formed hastily and poorly in the tumour, leading to poor blood flow and nutrient supply to the cancer cells. As the tumour grows ever larger, the hungry cancer cells become starved of nutrients.

For a tumour to grow and disease to progress, cancer cells must adapt to glucose starvation conditions. Cancer cells are adept at changing their behaviour to suit their conditions, helping them to thrive in this harsh environment. Revelations that βIII-tubulin interacts with proteins that help cells adapt to harsh conditions provided compelling support for the notion that βIII-tubulin makes cancers more aggressive and resistant to treatment by helping cancer cells adapt to the harsh conditions of the tumour environment.

To test this theory, we employed RNA interference and gene-editing techniques to manipulate the levels of the βIII-tubulin protein present in cancer cells and examine how these modifications affect the ability of cancer cells to grow and survive in an environment that mimics that of the tumour. We found that the high level of βIII-tubulin in cancer cells enhances the ability of cancer cells to adapt to glucose starvation conditions in the tumour environment, enabling them to grow and survive where other cancer cells cannot.

It achieves this by acting in different ways on multiple adaptation mechanisms. βIII-tubulin reduces the reliance of cancer cells on glucose to support their metabolic activity, allowing them to generate energy when glucose availability is limited.

In addition to being a fuel source, glucose is also used to generate chemical tags that help proteins to assume the correct folding pattern. When there is not enough glucose, proteins cannot be tagged so they fail to fold properly.

The endoplasmic reticulum is the protein-processing centre of the cell, and the site at which these glucose-derived tags are attached to proteins. When glucose is in short supply, misfolded proteins can accumulate in the endoplasmic reticulum, hampering cellular function and causing cells to stop growing until the burden of misfolded proteins can be reduced. If misfolded proteins keep accumulating in the endoplasmic reticulum over hours and days then the cell will die.

We found that βIII-tubulin helps to maintain the protein-processing capacity of the endoplasmic reticulum, thus preventing the accumulation of misfolded proteins when cells are challenged by glucose starvation. This enables cancer cells to continue to survive and grow in the glucose-starved tumour environment, when other cells would be crippled by a dysfunctional protein processing machinery. By having high levels of this protein, cancer cells are arming themselves against what is often a harsh tumour environment, allowing the tumours to grow more rapidly and aggressively.

New Cancer Treatments on the Horizon

These surprise findings provide the first functional evidence that βIII-tubulin makes cancer cells more resilient to the challenges raised by the tumour environment, and as a result contributes towards more aggressive tumours that are difficult to treat. This extends our understanding of the role of microtubule proteins in cell biology, challenging the prevailing perception of these proteins as purely structural entities.

However, there is much more to discover about the activities of βIII-tubulin and other microtubule proteins in cancer cells. We are now delving more deeply into how βIII-tubulin is functioning on a molecular scale to comprehensively define how this protein is integrated into protein networks that govern fundamental cellular behaviour. This approach is likely to reveal even greater complexity in the influence of microtubule proteins on basic cellular functions, and will prove critical in developing the next generation of anti-cancer agents.

By identifying that βIII-tubulin modulates glucose metabolism and protein processing in cancer cells, it’s now possible to develop treatment strategies incorporating the use of existing drugs that target these processes in cancer cells with high levels of this protein. Also, by exploiting the unique features of the microtubule cytoskeleton in cancer cells, such as the presence of the βIII-tubulin protein, these treatment strategies may selectively kill cancer cells while leaving the surrounding healthy cells unharmed. The trick will be ensuring that these selective treatments will be delivered to target the βIII-tubulin protein in cancer cells while staying away from nerve cells.

Nanoparticles carrying these selective drugs can be engineered to sail harmlessly through the bloodstream past nerve cells until they reach the poorly formed blood vessels of the tumour and become trapped, accumulating close to cancer cells and unloading their toxic cargo. Some of this toxic cargo may also spill onto the healthy cells that are direct neighbours of tumour cells, but because these cells lack the βIII-tubulin protein they will be unharmed. Promising developments in nano­particle design indicate that these technologies are now close to reality, paving the way for therapies that exploit both the internal and external features of the cancer cell’s environment.

These treatment strategies not only have the potential to more effectively treat cancer but also minimise the debilitating side-effects associated with existing chemotherapy treatments. These new horizons offer hope to cancer patients whose cancers have responded poorly to existing therapies, and once again place microtubules sharply in focus for the future of cancer treatment.

Amelia Parker completed her PhD thesis on the role of ßIII-tubulin in cancer progression with the Tumour Biology and Targeting Program at the Children’s Cancer Institute and the University of NSW, and is currently extending the findings of her study at the Children’s Cancer Institute.