For the first time, scientists from the German Cancer Research Center (DKFZ) and the National Center for Tumor Diseases (NCT) Heidelberg have characterised cancer cells that can initiate metastasis in the blood of breast cancer patients. These cells have properties of cancer stem cells and are characterised by three surface proteins. Patients with large numbers of these cells found in their blood show a rather unfavourable disease progression. The pattern of the three molecules may therefore be used as a biomarker for disease progression. The scientists plan to investigate whether the characteristic surface molecules may be used as targets for specific therapies for patients with advanced breast cancer.
Individual cancer cells that break away from the original tumour and circulate through the blood stream are considered responsible for the development of metastases. These dreaded secondary tumours are the main cause of cancer-related deaths. Circulating tumour cells (CTCs) detectable in a patient’s blood are associated with a poorer prognosis. However, up until now, experimental evidence was lacking as to whether ‘stem cells’ that lead to metastases can be found among CTCs.
‘We were convinced that only a very few of the various circulating tumour cells are capable of forming a secondary tumour in a different organ,’ says Prof. Andreas Trumpp, a stem cell expert. ‘Many patients do not develop metastases even though they have cancer cells circulating through their blood.’ Trumpp is head of DKFZ’s Division of Stem Cells and Cancer and director of the Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM) at DKFZ. ‘Metastasis is a complex process and cancer cells need to have very specific properties for it,’ he says. ‘Our hypothesis was that the characteristics of cancer stem cells, which are resistant to therapy and very mobile, are best suited.’

Irène Baccelli from Trumpp’s team developed a transplantation test for the experimental detection of metastasis-initiating cells. In collaboration with Prof. Andreas Schneeweiss from the National Center for Tumor Diseases (NCT) Heidelberg, along with colleagues from the Institute of Tumor Biology in Hamburg and the Institute of Pathology of Heidelberg University Hospitals, the researchers analysed the blood of more than 350 breast cancer patients. Using specific surface molecules, Baccelli isolated circulating tumour cells from the blood and directly transplanted them into the bone marrow of mice with defective immune systems. ‘Bone marrow is a perfect niche for tumour sells to colonise,’ Trumpp explains. After more than one hundred transplantations, metastases started forming in the bones, lungs and livers of some of the animals.

This proved that CTCs do contain metastasis stem cells – even though their frequency is apparently low. What characterises these cells? To define their molecular properties, the researchers analysed the surface molecules of the CTCs that had led to metastases after transplantation.

Three molecules characterise the metastasis stem cell
In a systematic screening process, Baccelli first isolated cells carrying a typical breast cancer stem cell protein (CD44) on their surface from the CTCs. This protein helps the cell to settle in bone marrow. Next, the researchers screened this cell population for specific surface markers which help the cells to survive in foreign tissue. These include, for example, a signalling molecule (CD47) that protects them from attacks by the immune system, and a surface receptor that enhances the cells’ migratory and invasive capabilities (MET).

Using a cell sorter, the researchers were then able to isolate CTCs which simultaneously exhibit all three characteristic molecules (CD44, CD47, MET). Another round of transplantation tests showed that these were in fact the cells from which the metastases originated.

Depending on the patient, cells exhibiting all three surface molecules (‘triple-positive’ cells) made up between 0.6 and 33 percent of all CTCs. ‘It is interesting that only cells with the stem cell marker CD44 carry the combination of the other two surface molecules,’ said Irène Baccelli. ‘It looks like the triple-positive cells are a specialized subtype of breast cancer stem cells circulating in the blood.’

Triple-positive cells as prognostic biomarkers

Are the triple-positive cells a more precise biomarker of breast cancer progression than the number of CTCs alone? In a small patient group, the researchers observed that as the disease advances, the number of triple-positive cells increases, but the total number of CTCs does not. In addition, patients with very high numbers of triple-positive cells had particularly high numbers of metastases and a much poorer prognosis than women in whom only a few metastasis-inducing cells were detected. ‘On the whole, triple-positive cells seem to have a substantially higher biological relevance for disease progression than previously studied CTCs,’ Andreas Schneeweiss explains. The researchers plan to confirm these new results in a large study.

Andreas Trumpp considers it good news that metastasis-initiating cells are characterised by the two proteins CD47 and MET. Therapeutic antibodies that target CD47 and inhibit its functions are already being developed. A substance inhibiting the activity of the MET receptor has already been approved and shows good effectiveness in treating a type of lung cancer. The substance may also help breast cancer patients with detectable metastasis-inducing cells. ‘The triple-positive cells we have found turn out to be not only a promising biomarker of disease progression in breast cancer but also a prospect for potential new therapeutic approaches for treating advanced breast cancer,’ says Andreas Trumpp. German Cancer Research Center

Cancer is typically thought to develop after genes gradually mutate over time, finally overwhelming the ability of a cell to control growth. But a new closer look at genomes in prostate cancer by an international team of researchers reveals that, in fact, genetic mutations occur in abrupt, periodic bursts, causing complex, large scale reshuffling of DNA driving the development of prostate cancer.
The scientists, led by researchers from Weill Cornell Medical College, the Broad Institute, Dana-Farber Cancer Institute and the University of Trento in Italy, dub this process ‘punctuated cancer evolution,’ akin to the theory of human evolution that states changes in a species occur in abrupt intervals. After discovering how DNA abnormalities arise in a highly interdependent manner, the researchers named these periodic disruptions in cancer cells that lead to complex genome restructuring ‘chromoplexy.’
‘We believe chromoplexy occurs in the majority of prostate cancers, and these DNA shuffling events appear to simultaneously inactivate genes that could help protect against cancer,’ says the study’s co-lead investigator Dr. Mark Rubin, who is director of the recently-established Institute for Precision Medicine at Weill Cornell Medical College and NewYork-Presbyterian Hospital/Weill Cornell Medical Center.
‘Knowing what actually happens over time to the genome in cancer may lead to more accurate diagnosis of disease and, hopefully, more effective treatment in the future,’ says Dr. Rubin, also the Homer T. Hirst III Professor of Oncology, professor of pathology and laboratory medicine and professor of pathology in urology at Weill Cornell and a pathologist at NewYork-Presbyterian/Weill Cornell. ‘Our findings represent a new way to think about cancer genomics as well as treatment in prostate and, potentially, other cancers.’
The discovery of ‘chromoplexy’ came after the research team worked collaboratively to sequence the entire genomes of 57 prostate tumours and compare those findings to sequences in matched normal tissue.
Co-lead investigator Dr. Levi Garraway, of the Broad Institute and Dana-Farber Cancer Institute, and his collaborators then tracked how genetic alterations accumulated during cancer development and progression. They used advanced computer techniques to identify periodic bursts of genetic derangements.
‘We have, for the first time, mapped the genetic landscape of prostate cancer as it changes over time,’ says Dr. Garraway, a senior associate member of the Broad Institute and associate professor at the Dana–Farber Cancer Institute and Harvard Medical School. ‘The complex genomic restructuring we discovered, which occurs at discrete times during tumour development, is a unique and important model of carcinogenesis which likely has relevance for other tumour types.’
Co-senior author Dr. Francesca Demichelis, assistant professor at the Centre for Integrative Biology at the University of Trento who also serves as adjunct assistant professor of computational biomedicine at Weill Cornell, worked with her collaborators to understand how widespread the DNA mutations and alterations seen in the tumours were across the cancer samples, and what that might mean in terms of cancer progression and, potentially, treatment. ‘Information about what alterations are common, and which aren’t, will most likely help guide us in terms of cancer drug use and patient response,’ says Dr. Demichelis.
The researchers also report that future targeted cancer therapy may depend on identifying complex sets of genetic mutations and rearrangements in each patient.
‘Every cancer patient may have individual patterns of genetic dysfunction that will need to be understood in order to provide precise treatment. Multiple drugs may be needed to shut down these genetic derangements,’ says Dr. Rubin. ‘Providing those tests now on every patient isn’t possible, but our study suggests that punctuated cancer evolution may occur to provide a subset of genes that offer a selective advantage for tumor growth. If that is true, we may be able to zero in on a limited number of genetic drivers responsible for an individual’s prostate cancer.’ Weill Cornell Medical College

One of 100,000 children is born with Menkes disease, a genetic disorder that affects the body’s ability to properly absorb copper from food and leads to neurodegeneration, seizures, impaired movement, stunted growth and, often, death before age 3. Now, a team of biochemistry researchers at the University of Missouri has published conclusive scientific evidence that the gene ATP7A is essential for the dietary absorption of the nutrient copper. Their work with laboratory mice also provides a greater understanding of how this gene impacts Menkes disease as scientists search for a treatment.

Humans cannot survive if their bodies are lacking the ATP7A gene, yet children can develop Menkes disease when the gene is mutated or missing. Previously, scientists did not have a good model to test the gene’s function or develop an understanding of the underlying causes of the disease symptoms. In his new study, Michael Petris, associate professor of biochemistry, was able to modify mice so that they were missing the ATP7A gene in certain areas of the body, specifically the intestinal track where nutrient absorption takes place.

‘These findings help us to understand where in the body the function of this gene is vital and how the loss of the gene in certain tissues can give rise to Menke’s disease,’ said Petris, who is a researcher in the Bond Life Sciences Center and holds an appointment in the Department of Nutrition and Exercise Physiology. ‘We want to continue to explore the underlying biology of Menke’s disease to determine where we should focus our research efforts in the future. If we know which organs or tissues are most responsible for transporting copper throughout the body, we can focus on making sure the gene is expressed in those areas. This disease is ideal for gene therapy down the road.’

Petris found that young mice missing the ATP7A gene in their intestinal cells were unable to absorb copper from food, resulting in an overall copper deficiency that mimics symptoms of Menkes disease in children. Petris says it’s vital to ensure that the developing newborns absorb enough copper during the neonatal period when the demand for the mineral is highest.

‘Copper is a little-appreciated but essential trace mineral in all body tissues,’ Petris said. ‘Cells cannot properly use oxygen without copper; it helps in the formation of red blood cells, and it helps keep the blood vessels, nerves, skin, immune system and bones healthy. Normally, people absorb enough copper through their food. However, in the bodies of those with Menkes disease, copper begins to accumulate at abnormally low levels in the liver and brain and at higher than normal levels in the kidney and intestinal lining.’

Newborn screening for this disorder is not routine, and early detection is infrequent because it can arise spontaneously in families, Petris said. Many times, the disease is not detected until the symptoms are noticed, and by that time, it can be too late for any aggressive treatments.

‘The clinical signs of Menkes disease are subtle in the beginning, so the disease is rarely treated early enough to make a significant difference,’ he said. ‘However, a single dose of copper injected into mice within a few days of birth restored normal growth and life expectancy. Early intervention was critical because treatment that began after symptoms developed wasn’t successful.’

Petris says that understanding the roles of copper in biology may have far-reaching health implications for the general population because copper underpins many facets of biology, including the growth of cancer tumours and the formation of toxic proteins in Alzheimer’s disease.

The development of these mice provides a novel experimental system in which to test treatments for patients with this disease. The early-stage results of this research are promising, but additional studies are needed. University of Missouri

A Johns Hopkins Children’s Center study in mice may help explain why women are more prone than men to a form of liver damage by implicating the female sex hormone oestrogen in the development of autoimmune hepatitis.
A life-threatening condition that often requires transplantation and accounts for half of all acute liver failures, autoimmune hepatitis is often precipitated by certain anaesthetics and antibiotics. Researchers say these drugs contain tiny molecules called haptens that ever so slightly change normal liver proteins, causing the body to mistake its own liver cells for foreign invaders and to attack them. The phenomenon disproportionately occurs in women, even when they take the same drugs at the same doses as men.
Results of the new study reveal that oestrogen and a signalling molecule called interleukin-6 collude to form a powerful duo that leads to immune cell misconduct and fuels autoimmune liver damage.
The findings, the research team says, also suggest therapeutic strategies to curb damage in people who develop drug-induced liver inflammation.
‘Our study shows that oestrogen is not alone in its mischief but working with an accomplice to set off a cascade of events that leads to immune cell dysregulation and culminates in liver damage,’ says Dolores Njoku, M.D., a pediatric anaesthesiologist and critical care expert at Johns Hopkins Children’s Center.
In the study, led by Njoku, researchers induced liver inflammation in mice by injecting them with drug-derived haptens. Female mice developed worse liver damage than male mice, and castrated male mice fared worse than their intact brethren, likely due to loss of testosterone and altered oestrogen-to-testosterone ratio, the researchers say. Female mice with missing ovaries — the chief oestrogen-secreting organs — suffered milder forms of hepatitis than mice with intact ovaries.
Female mice produced more liver-damaging antibodies and more inflammation-triggering chemicals, specifically the inflammatory molecule interleukin-6, known to fuel autoimmunity. Liver damage was notably milder in female mice whose interleukin-6 receptors were blocked or missing compared with normal female mice. On the other hand, male mice and female mice with missing ovaries had nearly undetectable levels of interleukin-6, while castrated male mice showed simultaneous upticks in both oestrogen and interleukin-6.
The research team further zeroed in on a class of cells known as regulatory T cells, whose main function is keeping tabs on other immune cells to ensure they don’t turn against the body’s own tissues. When researchers compared the number of regulatory T cells present in the spleens of male and female mice, they noticed far fewer regulatory T cells in the spleens of female mice. The spleen, the researchers explain, is the primary residence of mature immune cells.
‘Deficiency of regulatory T cells effectively takes the reins off other immune cells, leading to overactive immunity,’ Njoku says.
In a final, dot-connecting move, the researchers immersed spleen-derived immune cells in oestrogen. What they observed proved beyond doubt that oestrogen, interleukin and regulatory T cells form a powerful triangle. Oestrogen induced the immune cells of female mice to express more interleukin-6, which in turn diminished the expression of inflammation-taming regulatory T cells.
When the researchers injected sick female mice with a booster dose of regulatory T cells, their liver inflammation subsided to levels seen in male mice.
This powerful response, the researchers say, suggests that therapy with regulatory T cells may reduce
estrogen-related liver damage in patients with autoimmune hepatitis. Such treatment, however, remains years away from human application.
One reason, the researchers say, is that regulatory T cells maintain the fine equilibrium between overactive and underactive immunity. Because an overactive immune system can lead to autoimmune diseases and an underactive one can promote tumour growth, any therapy with regulatory T cells must be precisely calibrated to avoid tipping this precarious balance.
‘We first must figure out where the golden mean lies,’ Njoku says. John Hopkins Medicine

Researchers have uncovered mutations in the phosphatase Wip1 that enable cancer cells to foil the tumour suppressor p53. The results could provide a new target for the treatment of certain cancers.
Like a battlefield surgeon who has to decide which casualties can be saved, p53 performs triage on cells with injured DNA. If the damage is serious, p53 spurs the cells to die or stop proliferating. But after milder hits, p53 activates a DNA damage response (DDR) mechanism, which instigates repairs, and temporarily prevent cells from advancing any farther in the cell cycle. Once cells have mended their DNA, the phosphatase Wip1 enables them to re-enter the cell cycle by shutting down p53 and DDR proteins. Because p53 and the DDR stymie cancer cells, it’s no surprise that the rogue cells find ways to circumvent this protection. More than half of all cancers accrue mutations in the p53 gene, for example. Now, researchers from the Czech Republic and the Netherlands tested whether some cells instead carry mutations in the PPM1D gene, which encodes Wip1, to shut down p53.
The team analysed human tumour cell lines that harbour functional p53. Two of the lines displayed mutations in exon 6 of the PPM1D gene that resulted in a shortened version of Wip1. The truncated Wip1 was more stable than the full-length version of the protein, allowing cells to switch off p53 and continue the cell cycle in the presence of DNA damage. Depleting the truncated Wip1, however, halted the cell cycle until the DNA was repaired.
The researchers then looked for PPM1D mutations in 1,000 patients who had colorectal or breast and ovarian cancer. Four of the patients carried mutations, whereas none of the 450 cancer-free individuals did. All of these DNA alterations fell in exon 6 and caused production of shortened Wip1. To the researchers’ surprise, the mutations occurred in the cancer patients’ non-tumour cells as well. That suggests that the patients were born with PPM1D mutations, which set them up for cancer later in life but apparently caused no other illnesses.
‘We’ve identified a new mechanism that could lead to inactivation of p53 in cells and inactivation of the DNA damage response,’ says senior author Libor Macurek. The team suspects that PPM1D mutations could turn up in a variety of tumours. If so, targeting the short but overactive form of Wip1 could provide a new way to treat these cancers. EurekAlert