I was reading an article on MIT Technology review about superpatients for low cholesterol that got me thinking whether such patients exist for cancer.
The article is
I had also previously read an article on naked mole rats and the hyaluronan in the ECM.
Therefore, I am wondering whether there are any current studies, that is trying to discover the genetic outliers for cancers. Ie, humans that have hyaluronan in their ECM that is almost naked mole rat like. Since I understand that with a genetic pool of 7+ billion there has to be at least one outlier.
Pancreatic cancer – early detection, immune response, and infection-based resistance
Approximately 1.6 percent of men and women will be diagnosed with pancreatic cancer at some point during their lifetime. In 2014, an estimated 64,668 patients were living with the disease. The five-year survival for pancreatic cancer is 8.2% and it is projected to be the second leading cause of death due to cancer (behind lung cancer) in the US by the year 2030. For good reason, then, November is Pancreatic Awareness Month. Several recent research items are of particular interest to us. Continue reading &rarr
News Brief: A new source of drug resistance
The best available treatments for pancreatic cancer are highly toxic, and, as chemotherapies go, not very effective. The drug gemcitabine has been used for decades to extend the life of patients, but very high doses are required to combat the tumor, which grows in the pancreas surrounded by stiff, fibrous, noncancerous tissue called stroma. This hallmark of pancreatic cancer makes it unusually difficult to treat: the more stromal tissue accumulates, the less the drug works, while patients still endure brutal side effects. Only 8.5 percent of pancreatic cancer patients survive five years beyond their diagnosis, so there’s an urgent need to figure out why existing treatments are failing.
Scientists have known for a long time that gemcitabine fights cancer by killing cells during replication, though why it works for pancreatic cancer in particular is a bit of a mystery. The drug is a small molecule that masquerades as the nucleoside deoxycytidine, one unit in the nucleic acids that make up DNA. Once gemcitabine is integrated into a replicating strand of DNA, additional nucleosides can’t be joined to it. The new DNA strand can’t be completed, and the cell dies. Now, researchers from MIT have discovered that non-cancer cells in the pancreatic stromal tissue secrete astonishing quantities of deoxycytidine. They found that competition with deoxycytidine makes its imposter, gemcitabine, less effective, explaining why higher doses of the drug are needed as more stromal tissue grows around the tumor.
“That was an answer we were looking for — what is making pancreatic tumors resistant to gemcitabine?” says Michael Hemann, associate professor of biology, a member of MIT’s Koch Institute for Integrative Cancer Research, and co-senior author of the study. “Understanding the basic mechanisms of these drugs allows us to return to the clinic with improved strategies to treat patients with cancer.”
Douglas Lauffenburger, a professor of biological engineering, is also a co-senior author of the study, which represents a collaboration between the Hemann lab, the Lauffenburger lab, and the Vander Heiden lab, and appeared online in Cancer Research on September 4. Hemann lab graduate student Simona Dalin is the lead author.
The mystery ingredient
For years, researchers at MIT have been investigating different sources of chemotherapy resistance in stromal tissue. When Dalin took up the study two years ago, she was building on the findings of a former postdoc in the Hemann lab, Emanuel Kreidl. Kreidl had found that stellate cells, one type of cell in the pancreatic stromal tissue surrounding the tumor, were releasing something into the microenvironment of the pancreas that disrupted the function of gemcitabine.
Cells secrete all sorts of things — micro RNAs, fatty acids, proteins — that may be taken up and used by neighboring cells. Biologists call these ambient materials around the cell its “media.” Kreidl had tried boiling, digesting, and filtering the stellate cell media, but nothing he did made gemcitabine any more effective against the cancer cells. The usual suspects commonly implicated in drug resistance caused by neighboring cells, like proteins, would break down under such tests. “That’s when we knew there was something new here,” says Dalin. Her challenge was to figure out what that mystery ingredient was.
Mark Sullivan PhD ‘19, then a graduate student and biochemist in Vander Heiden lab, was enlisted to help separate the stellate cell media into its molecular components and identify them. After doing so, Dalin says, “it was fairly obvious that deoxycytidine was the thing that we were looking for.” Because gemcitabine works by taking deoxycytidine’s place in DNA replication, it made sense that the presence of a lot of deoxycytidine could make it difficult for gemcitabine to fulfill its function.
Molecules pass in and out of cells through gates in the cell membrane, called transporters. Using a drug that blocks certain transporters, Dalin was able to shut the gate in the stellate cells through which deoxycytidine is released. With less deoxycytidine around, the gemcitabine was effective at lower doses, confirming her hypothesis. Now, the researchers just needed to figure out how and where deoxycytidine was getting in the way of the drug.
Once inside the cell, a nucleoside must have one or more phosphate groups added to it by several enzymes in order to become a nucleotide that can be used to build DNA. Gemcitabine goes through the same process. The researchers determined that gemcitabine was competing with deoxycytidine for the first of those enzymes, deoxycytidine kinase. When they flooded the cell with that enzyme, gemcitabine didn’t have to wait in line for its phosphate groups — and could get into the DNA to work its fatal subterfuge.
Going forward, the Hemann lab aims to identify drugs that could inhibit the production of deoxycytidine and restore the tumor’s sensitivity to gemcitabine. Senthil Muthuswamy, an associate professor of medicine at Beth Israel Deaconess Medical Center who was not involved in the research, says this study provides “new and important insights” into how and why tumors develop resistance to gemcitabine. The findings, he adds, are “likely to have important implications for developing ways to overcome gemcitabine resistance in pancreatic cancer.”
The study’s findings may shed light on other cancer treatments that work similarly to gemcitabine. For every nucleoside, there are look-alike molecules, or analogs, that are used in cancer therapies. For example, the purine analog fludarabine is used to treat acute myeloid leukemia, another tenacious carcinoma. These generic drugs have been adopted through trial and error in the clinic, but scientists don’t fully understand why they are effective at the molecular level.
In theory, nucleoside analog drugs should work interchangeably every nucleoside is necessary in either the replication of DNA or RNA. In practice, though, these drugs are only effective for certain cancers. The MIT researchers speculate that the sheer amount of deoxycytidine being produced in the pancreas could suggest that pancreatic cells have a particular need for deoxycytidine that also makes them more responsive to its analogs — perhaps explaining why gemcitabine targets pancreatic cancer cells effectively.
“Understanding more about nucleoside biology, and more about which organs have high levels of which nucleosides, might help us understand when to use which chemotherapies,” Dalin says.
This study leaves the researchers with many questions about how and why nucleosides are produced in the body, a realm of basic biology that is still poorly understood. It’s generally assumed that cells only make nucleosides for their own internal use in DNA replication. But pancreatic stellate cells produce a lot of deoxycytidine, far more than they need for themselves, suggesting the excess nucleosides may serve some unknown purpose in neighboring cells. Although more experiments are needed to determine this mysterious purpose, the MIT researchers have some ideas.
“These extra nucleosides introduce a possibility that perhaps making deoxycytidine is a normal function of stellate cells in the pancreas, in order to provide building blocks for the cells around them,” says Hemann. “And that’s a real surprise.”
This work was funded in part by a David H. Koch Fellowship and the MIT Center for Precision Cancer Medicine.
Targeted therapies, immunotherapies, and improved chemotherapies are being developed to reduce the suffering and mortality that come from human cancer. Although these approaches, and in particular combinations of them, are expected to succeed eventually to a large degree, they all suffer one obstacle: Populations of replicating cells move away—typically in a high-dimensional space—from any opposing selection pressure they encounter. They evolve resistance. It is possible, however, to develop a precise mathematical understanding of the problem and to design treatment strategies that prevent resistance if possible or manage resistance otherwise. In this article, we present the fundamental equations that characterize the evolution of resistance. We provide formulas for the probability that resistant cells exist at the start of therapy, for the average number and sizes of resistant clones, and for the probability of successful combination treatment. We also demonstrate that developing new therapies that only maximize the killing rate of cancer cells may not be optimal, and that instead the parameters determining the fraction of resistant cells and their growth rate have a larger effect on the long-term control of cancer. These mathematical tools inform the search process for optimal therapies that aim to cure cancer.
A systems biology approach to overcome TRAIL resistance in cancer treatment
Over the last decade, our research team has investigated the dynamic responses and global properties of living cells using systems biology approaches. More specifically, we have developed computational models and statistical techniques to interpret instructive cell signaling and high-throughput transcriptome-wide behaviors of immune, cancer, and embryonic development cells. Here, I will focus on our recent works in overcoming cancer resistance. TRAIL (tumor necrosis factor related apoptosis-inducing ligand), a proinflammatory cytokine, has shown promising success in controlling cancer threat due to its ability to induce apoptosis in cancers specifically, while having limited effect on normal cells. Nevertheless, several malignant cancer types, such as fibrosarcoma (HT1080) or colorectal adenocarcinoma (HT29), remain non-sensitive to TRAIL. To sensitize HT1080 to TRAIL treatment, we first developed a dynamic computational model based on perturbation-response approach, to predict a crucial co-target to enhance cell death. The model simulations suggested that PKC inhibition together with TRAIL induce 95% cell death. Subsequently, we confirmed this result experimentally utilizing the PKC inhibitor, bisindolylmaleimide (BIM) I, and PKC siRNAs in HT1080.
Recent advancements in the molecular understanding of CRPC have given us a number of potential biological targets for the treatment of CRPC. Many of the pathways and targets covered in this review currently have agents that are undergoing clinical trials, and some are FDA approved for the treatment of CRPC. Unfortunately, most of these pharmaceutical agents only moderately increase survival and CRPC still remains incurable. However, recent discoveries and avenues of research may enable more effective molecularly targeted therapies as well as a better understanding of the mechanisms of CRPC.
Michael M. Gottesman, M.D.
Dr. Gottesman and colleagues pioneered the characterization of molecular mechanisms that result in failure to cure cancer with chemotherapy. They now focus on mechanisms that reduce accumulation of cytotoxic drugs in cancer cells, including reduced drug uptake and increased energy-dependent drug efflux by ABC transporters such as P-glycoprotein (ABCB1), ABCC1 (MRP), and ABCG2 (BCRP). They seek to define molecular tools to detect resistance mechanisms in cancers and devise ways to circumvent or exploit this resistance for treatment of multidrug-resistant cancers. An additional goal is to define the physiological role of these multidrug transporters in normal human tissues, e.g. at the blood-brain barrier.
1) multidrug resistance, 2) ABC transporters, 3) thiosemicarbazides, 4) blood-brain barrier, 5) chemotherapy
Success in treatment of some disseminated cancers with chemotherapy has led to intensified efforts to understand why many other cancers are intrinsically resistant to anticancer drugs or become resistant to chemotherapy after many rounds of treatment. Work in the Multidrug Resistance Section has revealed that a major mechanism of resistance of cancer cells to natural product anticancer drugs such as Adriamycin, etoposide, vinblastine, actinomycin D, and Taxol is expression of an energy-dependent drug efflux pump, termed P-glycoprotein (P-gp, encoded by the ABCB1 gene), or the multidrug transporter. This pump system contributes to drug resistance in about 50 percent of human cancers by preventing accumulation of powerful anticancer drugs in cancer cells. The sequence of the ABCB1 gene, determined in our laboratory, has led to (1) a model of the transporter as a pump with 12 transmembrane domains and two ATP sites, and (2) the discovery of a related family of 48 human ABC transporters involved in a variety of essential transport processes in cells. Polymorphisms in the ABCB1 gene, including the "silent" polymorphism C3435T (no amino acid change) affect drug resistance and sensitivity to inhibitors, probably by changing mRNA structure and the rate of translation. At least a dozen other ABC transporters may contribute to drug resistance in cancer. While the studies on the mechanism and function of P-gp and related ABC transporters in cultured cancer cells has led to a better understanding of possible mechanisms of multidrug resistance and novel ways to circumvent or target resistance such as the super-sensitivity of P-gp-expressing cells to some drugs, clinical relevance is still unclear.
One of the recognized roles for ABC transporters, especially P-gp and ABCG2, is that of limiting drug penetration into the brain. While knockout mouse models have been invaluable in defining the role of P-gp and ABCG2 at the blood-brain barrier, these models are expensive and not amenable to high-throughput assays. The zebrafish has been suggested as a possible alternative to the mouse model however, little data are available with regard to homologous transporters. Zebrafish do not have a direct homolog of ABCB1 but instead have 2 similar genes, abcb4 and abcb5. In addition, zebrafish have 4 homologs of human ABCG2—abcg2a, abcg2b, abcg2c, and abcg2d. We are currently localizing these transporters in the zebrafish and characterizing their substrate specificity to determine the feasibility of the zebrafish as a model for transporters at the human blood-brain barrier. Additionally, we are developing novel zebrafish-based reporter assays to model transporter inhibition at the blood-brain barrier.
Ongoing projects in the laboratory include genome-wide CRISPR screens to identify novel mechanisms of resistance to various chemotherapy agents in cell line models. In particular, we are interested in the mechanisms of resistance to platinum-based drugs and taxanes, as these are frequently used to treat ovarian cancer. Novel resistance mechanisms discovered from cell line models will be queried against a cohort of RNA Seq data from ovarian cancer tumor samples obtained after debulking surgery and at the time of relapse. Additionally, we seek to identify novel resistance mechanisms to oxaliplatin in colon cancer models, as oxaliplatin is one of the most effective drugs used to treat colon cancer. Finally, histone deacetylase inhibitors (HDIs) are particularly effective in T-cell lymphomas in the clinic, but relatively few resistance mechanisms have been reported. CRISPR screens identifying novel resistance mechanisms may lead to more responses in T-cell lymphomas when HDIs are combined with other targeted therapies.
While overexpression of ABCB1, ABCC1 or ABCG2 is known to confer a multidrug resistance phenotype, other ABC transporters may also contribute to drug resistance in cancer. We are therefore developing a CRISPR-based system to examine the role of all 48 ABC transporters in the development of resistance to a given chemotherapy. This system can be used with any cell line model of choice and with any cytostatic or cytotoxic compound.
Michael B. Yaffe, M.D., Ph.D.
Massachusetts Institute of Technology
Dr. Yaffe is the David H. Koch Professor of Biology and Biological Engineering at the Koch Institute for Integrative Cancer Research at the Massachusetts Institute of Technology. He is also a senior associate member of the Broad Institute, and an attending surgeon/surgical intensivist in the Departments of Surgery and Anesthesia at Beth Israel Deaconess Medical Center, Harvard Medical School. Dr. Yaffe received his B.S. in materials science and engineering from Cornell University, and his M.D. and Ph.D. degrees from Case Western Reserve University, followed by advanced postdoctoral training with Prof. Lew Cantley at Harvard Medical School. Dr. Yaffe’s research focuses on the biology of the complex signaling pathways that cells use to respond to DNA damage and inflammation, particularly the role of protein kinases and modular binding domains in tumor development and anti-cancer therapeutics. His laboratory uses a multidisciplinary approach encompassing systems biology, molecular pharmacology, biochemistry/proteomics, cell and structural biology, and computation/bioinformatics. Dr. Yaffe is also the scientific editor-in-chief of Science Signaling and a member of the editorial boards of Molecular and Cellular Proteomics, and Cell Cycle.
Jeffrey Engelman, M.D., Ph.D.
Harvard Medical School
Dr. Engelman is the principal investigator of his own laboratory at the Massachusetts General Hospital (MGH) Cancer Center, the director of thoracic oncology at MGH and the director of molecular therapeutics at the MGH Cancer Center. He received his B.A. in chemistry from Northwestern University and his M.D. and Ph.D. degrees from the Albert Einstein College of Medicine. Dr. Engelman completed his medical residency in internal medicine at Brigham and Women’s Hospital and his fellowship in hematology and oncology at the Dana-Farber Cancer Institute/MGH combined program. He joined the Harvard Medical School faculty and MGH in 2005. The research goal of his laboratory is to advance targeted therapies to benefit patients with cancer. His research focuses on understanding the biological underpinnings of sensitivity and resistance to specific kinase inhibitor targeted therapies in cancers with specific genetic abnormalities. In particular, his laboratory focuses on the regulation of key signaling networks that regulate cancer cell growth and survival. In his role as the director of thoracic oncology at MGH, he directs the research program of the thoracic oncology team. This program integrates laboratory studies, clinical trials, and comprehensive molecular analyses of cancers to pioneer individualized therapies. He has established a well-developed translational infrastructure that has culminated in a seamless bench-to-bedside connection with each activity informing the other.
Michael W. Deininger, M.D., Ph.D.
University of Utah
Salt Lake City, UT
Dr. Deininger is professor and chief of hematology and hematologic malignancies in the Department of Internal Medicine and the Huntsman Cancer Institute at the University of Utah. He also serves as senior director of transdisciplinary research at the Huntsman Cancer Institute. He has extensive experience treating patients with blood cancers and holds a particular interest in chronic myeloid leukemia and myeloproliferative neoplasms, a group of blood cancers related to leukemia. As a clinician-scientist with a translational research focus, Dr. Deininger is heading an extramurally funded research laboratory that is dedicated to the study of signaling pathways, drug resistance, and new molecular therapies in leukemia.
Annalisa VanHook, Ph.D.
Dr. VanHook studied biology as an undergraduate at Kenyon College and received her Ph.D. from the Department of Human Genetics at the University of Utah. She completed a postdoctoral fellowship at the University of California, Berkeley, supported by the Howard Hughes Medical Institute, in the field of evolutionary developmental biology. Dr. VanHook joined the staff of Science Signaling/AAAS in 2008, where she is currently web editor of Science Signaling.
Spalaxis resistant to chemically-induced cancer
To assess experimentally if Spalax is resistant to chemically-induced carcinogenesis, we treated animals from different rodent species according to the following protocols:
Spalax and C57BL/6 mice were treated with DMBA/TPA to induce skin cancer . Spalax animals developed skin lesions within 10 days (Figure 1A, upper middle panel). Histological examination of hematoxylin and eosin-stained tissue sections demonstrated skin necrosis involving the deep parts of the dermis, massive infiltration of the affected areas with neutrophil leukocytes, and ulcerated epidermis focally covered with fibrino-purulent exudates (Figure 1A, lower middle panel). The subcutaneous skeletal muscle and bone tissues were not affected, and no tumor was identified. The wounds completely healed within seven to nine weeks, resulting in epidermal thickening (Figure 1A, right panels), and no further progression to skin tumors was observed, even though TPA treatments were extended to six months (November 2010 to April 2011). In the control group, Spalax animals treated with acetone only did not show any changes in their skin macro- and microstructure, similar to non-treated animals (Figure 1A, left panels). Following 7 to 10 days of DMBA/TPA treatment, mice demonstrated small intra-epidermal blisters some of them ruptured, forming superficial erosions with extensive crusting (Figure 1B, middle panels), which subsequently underwent transformation into multiple skin tumors within two to three months (Figure 1B, upper right panel). Histological examination revealed papillary and flat epidermal outgrowths with dysplastic features, focally similar to squamous cell carcinoma (Figure 1B, right panels).
Effect of DMBA/TPA carcinogenic applications on Spalax and mice skin . Macroscopic and microscopic skin changes in Spalax (A) and mice (B). (A) Normal tissues (left images). Necrosis of skin and subcutaneous adipose tissue (middle images). Completely healed skin lesion showing epidermal thickening with hyperkeratosis and dermal fibrosis (right images). Hematoxylin and eosin staining, ×40 (left and middle images) and ×100 (right image). (B) Normal tissues (left images). Intra-epidermal blisters, partially ruptured with erosion formation and crusting, congestion and inflammatory cell infiltrate within the dermis indicate ongoing inflammation (middle images). Skin papillary outgrowths with thickened, dysplastic epidermis, numerous mitoses and foci are suggestive of squamous cell carcinoma (right image). Hematoxylin and eosin staining, ×40 (left and middle images) and ×100 (right image). DMBA/TPA, 7,12-Dimethylbenz(a) anthracene/12-O-tetradecanoylphorbol-13-acetate.
The ability of a single subcutaneous 3-MCA injection to induce fibrosarcoma is well documented . The expected tumors appeared within two to three months in mice, and in four to six months in rats. Hypercellular spindle cell tumors with highly pleiomorphic, extensively proliferating cells (30 and more mitotic figures per 10 high power fields) arranged into intersected bundles or wide sheets were identified. Scant, partially myxoid stroma and areas of hemorrhagic necrosis were typical findings (Figure 2A). All examined tumors developed in 3-MCA-treated mice and rats were histologically identified as fibrosarcomas. Importantly, Spalax did not show any pathological process for over a year. However, by 14 to 16 months following the 3-MCA treatment, 2 of the Spalax animals (out of 6 old individuals and a total of 12 animals) developed a tissue overgrowth at the site of the injection. These lesions were well circumscribed in shape, unlike the ill-defined tumors found in mice and rats (Figure 2B). Histological examination revealed benign spindle cell proliferation most probably reflecting fibrosis at the site of an incompletely resolved inflammatory reaction.
Effect of 3-Methylcholantren treatment on soft tissue tumor induction in Spalax and mice. Animals treated with a single injection of 3MCA as described in the Materials and methods section. Representative images show macroscopic and microscopic observations. Mice (A): An ill-defined, soft mass, with foci of necrosis and hemorrhage diagnosed as high-grade fibrosarcoma by histology. Spalax (B): a well-circumscribed, firm, whitish nodule composed of benign spindle cells organized into long regular bundles - benign reactive fibrosis. Hematoxylin and eosin staining, ×100. 3MCA, 3-Methylcholantrene.
A case of fibrosarcoma development in Spalax
A single, old Spalax individual developed a 3-MCA-induced tumor 18 months after initial treatment (Figure 3). A biopsy was performed, and the histological examination revealed a partially necrotic and heavily inflamed, spindle and epithelioid cell tumor with infiltrative borders and myxoid stroma. Cells demonstrated dyscohesion, polymorphism in size and shape (bizarre and giant cells present) and prominent nuclear atypia (Figure 3A). This hypercellular tumor demonstrated high mitotic activity (above 30 mitoses per 10 high power fields) with abundant atypical mitotic figures. Transmission electron microscopy revealed fibrosarcoma-like findings : deformed nuclei, some with monstrous appearance long branching and dilated rough endoplasmic reticulum and abundance of extracellular collagen fibers (Figure 3B,C). Myofibroblastic differentiation features were not observed. An immortal cell line was established from the tumor sample. The cultured adherent cells show a typical fibroblast phenotype (Figure 3D), which has remained unchanged throughout a long culture time (40 passages, 8 months after isolation).
3MCA-induced tumor in Spalax . (A) Light microscopic examination. Note spindle, epithelioid and giant multinuclear cells (empty arrow) nuclei are variable in shape, size and chromatin distribution nucleoli vary in frequency. Hematoxylin and eosin staining, ×100. (B) Transmission electron microscopy (TEM): dilated, elongated rough endoplasmic reticulum (black arrows) and abundant collagen fibers (white arrows) (C) TEM: a giant, monstrous nucleus (N). (D) Cell line established from Spalax tumor, phase contrast image after six months of continuous cultivation (×200). 3MCA, 3-Methylcholantrene.
The remaining treated Spalax individuals showed no phenotypic or behavioral changes, and were still under observation in the Animal House over two years following treatment (October 2010 to July 2013).
Spalax fibroblasts suppress growth of human cancer cells in vitro
To compare the effects of normal fibroblasts isolated from different rodents on the growth of human cancer cells, we used a co-culture approach, where fibroblasts were cultured together with cancer cells on a shared surface (Figures 4 and 5). In these experiments, hepatoma-derived Hep3B cells as well as breast cancer MCF7 cells were tested. Obvious inhibition of cancer cell growth was found when Hep3B cells were co-cultured with Spalax normal lung and skin fibroblasts: the foci of destroyed cancer cells were visible after six days of co-culture (Figure 4). Prolonged co-cultivation up to 11 days resulted in further destruction of cancer cell colonies by the presence of Spalax fibroblasts and the spaces previously occupied by Hep3B cells were invaded by fibroblasts (Figure 4). In contrast, the number of cancer cells co-cultured with mouse fibroblasts increased gradually, and on Day 6, Hep3B cells surrounded by mouse fibroblasts reached approximately 80% confluence, similar to control (Hep3B only). Overgrown Hep3B colonies were found after 11-day co-culture with mouse fibroblasts. An obvious inhibitory effect was demonstrated when Spalax normal skin fibroblasts were co-cultured with breast cancer MCF7 cells as well (Figure 5). After 10 days of co-culture with Spalax fibroblasts, massive rounding and detachment of cancer cells were observed. On the other hand, mouse fibroblasts stimulated proliferation of MCF7 cells, and by Day 10 densely populated colonies of cancer cells developed.
Effects of Spalax and mouse fibroblasts on growth of co-cultured human hepatoma cells. Tumor cells (TC) were cultured either alone or in the presence of Spalax or mouse fibroblasts in the ratio of 1:10 (5 × 10 4 fibroblasts and 5 × 10 3 cancer cells in six-well plates) in RPMI/DMEM-F12 media (1:1) containing 10% FBS. White arrows point to the foci of destroyed cancer cells, and black arrows show the fibroblast-tumor cell colony boundaries. Cells in mono- and co-cultures were observed and photographed daily. Representative images for each sample at different time intervals are shown (×200).
Morphologic alterations in human breast cancer MCF7 cells triggered by co-culture with Spalax fibroblasts. MCF7 cells were co-cultured with skin fibroblasts of Spalax or mouse in the ratio of 1:15 (5 × 10 4 fibroblasts and 2.5 × 10 3 cancer cells in six-well plates) in DMEM/DMEM-F12 media (1:1) containing 5% FBS. Representative phase contrast images after 10 days of co-culture are presented (×200). Note rounding and detachment of MCF7 cells co-cultured with Spalax fibroblasts. Black arrows point to rounding cells. White arrows show shrunken “floating” cells.
In vitroanticancer activity by other wild, natural rodents’ fibroblasts
Since we compare a wild mammal with laboratory animals that are sensitive to cancer, we conducted co-culture experiments using Hep3B cancer cells with skin fibroblasts isolated from two different wild, natural rodents: Acomys, a short-lived, wild, above-ground rodent and naked mole rat (Heterocephalus glaber), a long-lived cancer-resistant wild subterranean rodent . As shown (Figure 6), no growth inhibitory effect was found when Acomys fibroblasts were co-cultured with Hep3B cells. On the contrary, Acomys fibroblasts promoted cancer cell invasion similar to the effect of rat fibroblasts. Heterocephalus cells, similar to Spalax, evidently destroyed cancer cell growth (Figure 6).
Comparison of the effects of Spalax, Acomys, Heterocephalus and rat skin fibroblasts on growth of Hep3B cells. Hep3B tumor cells were cultured either alone or in presence of Spalax, Acomys, Heterocephalus or rat fibroblasts in the ratio of 1:10 (5 × 10 4 fibroblasts and 5 × 10 3 cancer cells in six-well plates) in RPMI/DMEM-F12 media (1:1) containing 10% FBS. After seven days incubation cells were photographed. Representative images for each sample are shown (×200). White arrows point to the foci of damaged cancer cells. TC, tumor cells.
Conditioned medium generated by Spalaxfibroblasts induces cancer cell death, but does not affect normal primary fibroblasts
To determine whether the anti-cancer activity of Spalax fibroblasts was mediated by fibroblast-secreted soluble factors, conditioned media (CM) obtained from Spalax, mouse and rat monolayers were tested. Cancer cells of different origins were incubated under CM of normal fibroblasts, which had never been exposed to cancer cells or other stimuli. Effects of CM generated by cancer cells were also tested (Figure 7). As demonstrated in Figure 7A, exposure of Hep3B cells to CM from cultured newborn Spalax fibroblasts decreased cancer cell viability as measured by mitochondrial respiratory function. Exposure to mouse CM hardly had an effect on cancer cell viability. Similarly, nine-day exposure of Hep3B cells to CM generated by adult (>5.5 years old) Spalax fibroblasts obviously reduced cancer cell viability as was determined by a trypan blue extrusion assay (Figure 7B,C): cancer cells exposed to Spalax fibroblast-conditioned CM reached 49% death, whereas unexposed cells remained completely adherent and viable (Figure 7C).
Effects of conditioned media (CM) on viability of cancerous and non-cancerous cells. (A) Hep3B cells were seeded in a 96-well plate at a density of 5 × 10 3 and 1 × 10 3 cells/well in RPMI-DMEM/F12 medium conditioned by Spalax or mouse skin newborn fibroblasts (SpNbF and MNbF, respectively). Hep3B cells were incubated for four days viability was estimated by PrestoBlue® Reagent. (B,C) Hep3B cells (1 × 10 4 cell/well) were cultured in six-well plates under conditioned medium of Spalax adult skin fibroblasts (B) or grown in medium generated by Hep3B cells (C). After nine days, the cells’ survival rates were assessed by a Countess® cell counter (Life Technologies) red: dead cells, blue: viable cells. (D) Hep3B and HepG2 cells were incubated under Spalax CM for four days, followed by changing the media either to fresh media or new Spalax CM. After two days, viability was estimated by PrestoBlue® Reagent. (E) Spalax fibrosarcoma cells (SpFS2240) were incubated for three or seven days in full medium or under CM of Spalax adult skin normal fibroblasts (SpAdF CM), Hep3B (Hep3B CM), Spalax fibrosarcoma (SpFS2240 CM). Cell viability was evaluated by using PrestoBlue® reagent. Results are presented as percentage of control (SpFS2240 CM) mean ± S.D. (F) Effects of CM generated by Spalax or mouse normal fibroblasts (SpNbF CM and MNbF CM, respectively) on the growth of non-cancerous cells. The viability was estimated after four days by PrestoBlue® reagent mean ± S.D. (G) Heat treatment of conditioned media. Seven-day CM, generated by Spalax or rat fibroblasts, was heat-treated at 56°C for 10 minutes and 30 minutes prior to addition to Hep3B cancer cells (2,000 cell/well) in 96-well plates. Cells were incubated for seven days followed by PrestoBlue® test. All results were obtained from three independent experiments performed in three to six technical repeats.
We next evaluated the reversibility of the inhibition of cancer cells initiated by Spalax CM. HepG2 and Hep3B were grown with Spalax CM for four days, then the medium was changed by either fresh unused regular media or with fresh Spalax CM. Cancer cell viability was measured after another two days. Recovery of the cancer cells was demonstrated when the CM was changed with fresh unused regular media (Figure 7D). Importantly, growth of Spalax-derived fibrosarcoma cells (SpFS2240) was gradually suppressed by CM generated by Spalax normal fibroblasts, but was not affected by normal, full medium and CM derived from Hep3B cells or CM derived from the SpFS2240 cells themselves (Figure 7E). Noteworthy, no inhibitory effects were detected on mouse, rat and Spalax normal fibroblasts following exposure to homologous or heterologous CM (Figure 7F). To get a preliminary idea of the nature of the secreted factors responsible for cancer cell growth inhibition, CM from Spalax and rat fibroblasts, and the regular medium of fibroblasts (DMEM-F12) were heated to 56°C for 10 minutes, and 30 minutes. The different heat-treated media was mixed 1:1 with RPMI (the optimal growth medium for the hepatoma cell lines used in this study) and was added to Hep3B cancer cells. After seven days, the viability of the cancer cells was measured. The heat-treated CM generated from Spalax fibroblasts reduced its anticancer activity, expressed as a partial increase in Hep3B cells viability (Figure 7G).
Soluble factors generated by Spalaxfibroblasts cause cell cycle arrest, nuclear fragmentation, and impair mitochondrial dynamics in cancer cells
To investigate the mechanisms by which Spalax fibroblasts induce cancer cell death, we examined nuclear and mitochondrial shape dynamics, as well as cell cycle distributions in Hep3B and HepG2 cells. No changes in the morphology of cells, nuclei and mitochondria as well as in cell cycle distribution were found when Hep3B cells were incubated with rat CM (Figure 8, middle row) compared to Hep3B grown with their own medium (Figure 8, upper row control). In contrast, following exposure to Spalax CM, Hep3B cells undergo phenotypic changes observed under phase contrast microscopy: cellular shrinkage, irregularities in the plasma membrane and blebs formation (Figure 8, lower row, phase-contrast). Cell cycle analysis revealed a noticeable accumulation of dead cells in sub-G1 (36.7% versus 16.4% in control), a reduction in the number of cells in G0/G1 (28.9% versus 49.6% in control), and a modest arrest of proliferation in G2/M (21.7% versus 17.1% in control) (Figure 8, lower row, cell cycle). Nuclear staining with DAPI of Hep3B cells that were grown with Spalax CM for eight days, revealed heterogeneous chromatin appearance within irregularly shaped nuclei, and in many cells extensive chromatin condensation and nuclear fragmentation were conspicuous (Figure 8, lower row, DAPI staining). On the other hand, homogeneous patterns with regular-shaped nuclei were mainly represented in the cells incubated with rat CM as well as in the control cells (Figure 8, upper and middle row, DAPI staining). To examine whether Spalax fibroblast CM could induce mitochondrial dynamic changes in cancer cells, Hep3B cells were stained with MitoTracker-Red® probe after eight days of incubation. Compared with control and rat CM, the mitochondrial network of cells after eight-day growth with Spalax CM demonstrated the presence of damaged fragmented mitochondria (Figure 8, lower row, MitoTracker® + DAPI). Similar to Hep3B cells, HepG2 cells under Spalax CM also showed morphological changes and accumulation of cells in sub-G0/G1 whereas mouse and rat CM did not affect cellular morphology and cell cycle distribution (Figure 9). BrdU incorporation into DNA, a marker for cell proliferation, confirmed a time-dependent anti proliferative effect of Spalax CM on HepG2 cancer cells (Figure 9E).
Spalax fibroblast-conditioned medium compromises cell cycle, causes nuclear and mitochondrial fragmentation in Hep3B cells. Hep3B cells were grown on cover slips under medium conditioned by Spalax or rat fibroblasts for seven days. Representative phase-contrast images demonstrating morphological changes (×200) are depicted. Cells were harvested and stained with PI, and cell cycles were analyzed by flow cytometry. Representative flow cytometry histograms of three independent experiments performed in duplicate are presented. Hep3B cells were stained with MitoTracker®Red, fixed with formaldehyde and counterstained with DAPI. Representative fluorescence microscopy images demonstrating nuclear and mitochondrial changes are present. White arrows point fragmented nuclei empty arrows point chromatin condensation. Scale bars represent 10 μm. PI, Propidium iodide.
Effects of Spalax , mouse and rat conditioned media on morphology and cell cycle progression in HepG2 cells. HepG2 cells were incubated under conditioned media for eight days thereafter, cell morphology was documented using phase contrast microscopy, harvested, stained with PI and analyzed by flow cytometry. Representative images (×200) and flow cytometry histograms are presented: (A) control media (B) rat CM (C) mouse CM (D) Spalax CM (E) BrdU incorporation assay: HepG2 were grown in 96-well plates (2000 cells/well) for four and seven days under Spalax-generated CM. BrdU Cell Proliferation ELISA (Exalpha) was used. Time-dependent decrease in cell proliferation under Spalax-generated CM is depicted. CM, Conditioned media PI, Propidium iodide.
Spalax normal fibroblasts inhibit colony formation in soft agar of the breast carcinoma cell lines MDA-MB-231 and MCF7 as well as Spalax-derived fibrosarcoma
To study whether soluble factors generated by Spalax fibroblasts may influence colony formation in soft agar, breast cancer cells were cultivated for three weeks in the absence or presence of Spalax fibroblasts (Figure 10). Spalax fibroblasts strongly reduced the formation of MDA-MB-231 colonies (Figure 10A,B). The ability of MDA-MB-231 to form large colonies was completely inhibited by Spalax fibroblasts (Figure 10C), while rat fibroblasts had no effect on colony formation (Figure 10A,B). Cells from another human breast cancer cell line, MCF-7, were incubated with monolayers of Spalax and mouse fibroblasts (Figure 10D). Remarkably, after 11 days, and compared to the control, more colonies were formed when human MCF7 cells were co-cultured with mouse fibroblasts, whereas a monolayer of Spalax fibroblasts significantly reduced MCF7 colony-formation.
Spalax fibroblasts suppress colony formation of human breast cancer cells MDA-MB-231 and MCF7 in soft agar. (A) MDA-MB-231 cells (5 × 10 3 cells) cells were suspended in 0.35% agar and added as the cancer cell top layer to base layer either empty (blank) or containing the Spalax or rat fibroblast monolayer. At Day 21, colonies larger than 50 μm were counted under an inverted microscope and photographed (×40). Representative microscopic images out of 15 fields are shown. (B) Average number of colonies counted in soft agar (n = 15). The experiment was performed in duplicate plates at least three times mean ± S.D. (C) A representative colony in soft agar was formed by MDA-MB-231 only, or by co-culturing with a Spalax fibroblast monolayer. The size bar shows equivalent magnification in both images (× 200). (D) MCF7 cells (5 × 10 3 cells) were grown in soft agar on top of a monolayer of mouse newborn (MNbF), or Spalax newborn fibroblasts (SpNbF) in 35-mm culture dishes. After 5 and 11 days of incubation colonies containing >20 cells were counted by using an inverted microscope (× 200), mean ± S.D.
Importantly, Spalax normal fibroblasts suppressed growth and colony formation of the homologous tumor, Spalax-derived fibrosarcoma (SpFS2240) (Figure 11). In contrast, both rat and mouse normal fibroblasts stimulated growth of Spalax tumor cells in soft agar (Figure 11A). Integrating the number of colonies and their total occupied area, calculated from five independent fields, revealed a 36% reduction when SpFS2240 were grown above a Spalax fibroblast monolayer compared to blank plates (Figure 11B, 2240 alone). In contrast, mouse and rat fibroblasts enhanced colony formation by factors of 1.7 and 2.1, respectively, compared to the blank plates (Figure 11B).
Effect of Spalax, rat and mouse fibroblasts on Spalax -derived fibrosarcoma cells colony formation. (A) SpFS2240 Cancer cells were grown in soft agar on top of monolayers of mouse, rat and Spalax fibroblasts. After three weeks, colonies were counted. At least 10 fields were recorded for each observation. Two representative images demonstrating effects of different fibroblasts on colony-formation are shown (×40). (B) Colony numbers and cumulative total colony area (μm 2 ) from five fields were calculated to demonstrate the effects of the fibroblasts monolayer on the cancer cell colony formation and growth.
Investigating Breast Cancer: The Underlying Biology of Drug Resistance
Advances in cancer therapy have dramatically contributed to the decline in breast cancer deaths over the last three decades. But even with these advances, drug resistance—when tumors stop responding to anti-cancer drugs—remains a serious clinical challenge. So how exactly do cancer cells evade the drugs designed to kill them? What's next in developing strategies to prevent or overcome drug resistance and improve outcomes in breast cancer patients? And what role can new technologies like liquid biopsies play?
In this episode of BCRF’s Investigating Breast Cancer, we talk to Dr. Sarat Chandarlapaty to answer these questions. Dr. Chandarlapaty is a laboratory head at the Human Oncology and Pathogenesis Program at the Memorial Sloan Kettering Cancer Center. He's also a BCRF Scientific Advisory Board member and has been a BCRF researcher since 2015.
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Read the transcript below:
Chris Riback: Dr. Chandarlapaty, thank you for joining me. I appreciate your time.
Dr. Sarat Chandarlapaty: Thank you, Chris. It's a pleasure to be here.
Chris Riback: Of course, I want to talk with you about resistance to therapy and the progress that you are making, that can be made, that you hope to make in those areas, but given our times, I think I should start and would love to start with a very brief Coronavirus update and really just in terms of what are you hearing? What are you hearing from the breast cancer community and what are you hearing from your patients?
Dr. Sarat Chandarlapaty: Yes, Chris, this is obviously an unprecedented time. As an oncologist, I think, I get these phone calls, really there are two sort of streams of questions. One is, "What can I do to avoid getting Coronavirus? I have breast cancer and I don't want to get Coronavirus," and then on the other side is, "Are we ignoring my breast cancer?" The answer to those, in some ways, competing questions is: "We're here to care for your breast cancer and to treat it as the disease that it is, but to recognize there's this unprecedented risk out there, and we do your care in a way that's tailored to this moment." But we're still very much in the business of trying to make sure that we offer the very best treatments for breast cancer.
Chris Riback: Let's talk about those treatments and let's talk about your research in particular. So your area of research I have seen described as solving the mysteries of drug resistance and improving response to targeted therapies. Is that how you think of it? Are you trying to solve a mystery?
Dr. Sarat Chandarlapaty: Yes, it's interesting, I mean, one of the first things we want to figure out about cancer is how can we cure it, how can we treat it and make those tumors shrink? And with breast cancer, we've solved a little of that problem, right? We've developed therapies over many years through the work of many, many people. We've developed some basic understandings of what makes some breast cancers tick and when I've come in, I've seen that and the question I struggle with is why does it work and then stop working? What is happening there?
Chris Riback: Yes.
Dr. Sarat Chandarlapaty: Because if we could just make those treatments work indefinitely, then I think we would have a far better solution. Yes, resistance lies at the heart of the research that I do.
Chris Riback: What defines or describes resistance to therapy? So for a lay person like me, we always hear about resistance, generally, I'll hear it in terms of antibiotics. "Don't take too many antibiotics, because you'll increase resistance to their effectiveness." Resistance in a tumor, resistance in breast cancer is something different.
Dr. Sarat Chandarlapaty: That's right. There are similarities to antibiotic resistance, but I would start by saying, there are two classes. First, there are cancers that we give a treatment to and those cancers clearly don't care. They just sort of proceed on as though we didn't treat them. That's a sort of intrinsic resistance and that's less common overall. Then there is the so called acquired resistance, that is a cancer that for six months, for three years, was treated with a drug and seemed to be well behaved under that regime, maybe shrunk some, and then suddenly started to grow, started to go into new places. That change in behavior is really the resistance that my lab has really focused in on, because it's so common— such a common occurrence for patients who have had, particularly, the more advanced breast cancers.
Chris Riback: How common is common? And I found myself wondering, are there any signs in advance, any commonalities where you kind of getting a hint that resistance might occur. So I guess, let's start with the beginning part, which is how common is it?
Dr. Sarat Chandarlapaty: Yes, so we think about it mainly in the setting of where patients have advanced disease, so called stage IV breast cancer, where cancer has spread outside of the breast, and we're predominantly treating with drugs, oral drugs, IV drugs.
Chris Riback: But has it metastasized because the resistance was there? Meaning, if the resistance didn't occur, it wouldn't have gotten to the state that you just described? And I know metastasis can occur for all sorts of reasons but when you're looking at it, are you talking about the type of metastasis that has occurred because there was a resistance in the first place or is there now resistance now that you're finding the cancer in the different organs?
Dr. Sarat Chandarlapaty: That's a good question and a little complicated. If a cancer presents for the first time, as a cancer that's not just in the breast but is in the breast and, say, the bone, that's a cancer that's never been exposed to a therapy, so it may have that so-called intrinsic resistance, but it certainly wouldn't have acquired resistance. It didn't get exposed to a therapy and change or adapt or evolve, but sometimes we actually do see that. We see a patient who presented with a primary breast cancer, had it removed surgically, received hormone therapy, for instance, and after years on hormone therapy, a breast cancer arises in a new site. And that's one that is resistant, did the resistance fuel its spread?
Dr. Sarat Chandarlapaty: Probably not but we don't know if sometimes the resistant cancer takes on new properties that allow them to spread, but I tend to think of this, to answer more simply, as separate processes. The cancer spreads and the cancer that has spread is resistant .
Chris Riback: To therapies at that point?
Dr. Sarat Chandarlapaty: To therapy, right.
Chris Riback: And getting back, I think I might have cut you off in terms of how common is it, because I would assume that this is an area of concern for someone and I want to ask you about that in a moment, but how common is it?
Dr. Sarat Chandarlapaty: For cancers that have spread, that are stage IV or metastatic, most of them, I would say, on the order of 80 to 90 percent will eventually figure out and become resistant to the therapy we give. The timing of that is quite variable, and remarkably so. So for one patient, on a very common regimen of a hormone and a targeted therapy combined, one person, their cancer might respond and then develop resistance in six months, and another person treated with the same regimen with the same characteristics might respond and then develop resistance six years later. So one is six months, one is six years and that's obviously, there are some intrinsic property is different about those cancers and we want to understand that.
Chris Riback: And are there signs or is it like a light switch? Is that resistance gradual and from your perch, you can, you see it coming or is it sudden and all of sudden, one day it's working and the next day it's not?
Dr. Sarat Chandarlapaty: Well, that's a really important question, whether we can develop technologies that can tell us when it's coming so that we can sort of be ahead of it. Right now, the standard way that we find this out is because we do serial imaging and blood tests like what we call tumor markers. Or we sort of listen to the patient for what symptoms might be happening and so it's somewhat crude that after three or four months, we'll see if the treatment's working. So things might be happening in a much earlier time point but we don't have ready access necessarily to technologies that can tell us about that, but if we could find it out earlier when it's just a few cells as opposed to a large number, then that may enable us to develop treatments that work better for the resistant cancers.
Chris Riback: Listening to you, I find myself, the word that keeps coming to my mind is uncertainty, and I'm thinking about kind of the emotional challenge of that. I'm imagining that you're working with patients who have already gone through what they have gone through and who like any of us would be looking for something that resembles . I put the word in quotes, "control" or "certainty," we all seek that in our lives and we probably can never have that, as I think maybe even this current pandemic is showing us. Having control over life is pretty tough to get, but I assume that's the goal but then there's this uncertainty that a certain percentage of cases, this resistance occurs and then there's the added uncertainty that the timing can be different. It's just emotionally, I would think this has to be something of a challenging area. Am I kind of imagining the situation correctly?
Dr. Sarat Chandarlapaty: No, I think you're right that we want to be able to have some understanding of the processes that are happening and not just that they're happening, but when they're happening and be able to plan accordingly and to have control. I agree, and having, I think, measures of what's happening, that are telling us about . in more detail whether someone is more likely to be, have a cancer that's in the type that's likely to develop resistance in six months versus 10 years can be helpful, particularly to the one who's in the 10 year group, right? And also gives us tools to be able to give us the insight that we might want to do something different for those that are more likely to be six months kind of group. I think understanding better that being able to make it a little more granular, I think is helpful for patients and it's helpful for obviously physicians as well.
Chris Riback: Now, this I assume is one of the hard parts in the quintessential $64,000 question which is why some tumors would be resistant. Maybe this is obvious but in looking at your research, and looking at the work that you've done, so I realize potentially, and maybe this is just simple question and I just wasn't getting it. But, for example, in the ER-positive patients, if the aromatase inhibitors, or that the inhibitors that prevent the production of estrogen and thereby starve the cancer of the fuel that ER-positive patients, that defines that, how does that cancer metastasize if it doesn't actually have the fuel that it needs to grow? I mean, I guess, is that the core of the question that you're trying to discover?
Dr. Sarat Chandarlapaty: Yes, what is it exactly that makes the cancer tick, and what allows it to start ticking again? You know, the two things that I think we've elaborated better is, first, that when the cancers for the ER-positive cancers that are treated with hormone therapy, and this is what we've worked a lot on, when they become resistant, they don't suddenly turn into a cancer that resembles a melanoma or a lung cancer, and starts looking for other sorts of fuel if you will. They actually try to reactivate that hormone program and the way they do that is just by developing mutations in the DNA, very specifically for those genes that work on estrogen program. And so they're addicted, in a way, to this program, and they try to reactivate it, rather than trying to turn themselves into something completely different.
Perhaps that's surprising but that's what, by learning that, we've then developed new drugs that can target that pathway in different ways. So if one hormone therapy doesn't work and it's because of mutation in the hormone pathway, then we can potentially use another drug, specifically in the hormone pathway, and that will work again. So it's targeting that core addiction of the cancer.
Chris Riback: I'm curious about liquid biopsies and I know many folks are curious about liquid biopsies. What should folks understand about how they work and how they should think about them or potentially could think about them in their own situations?
Dr. Sarat Chandarlapaty: Yes, it's a great question. This is a new area and didn't exist really 10 years ago. The idea that tumors secrete stuff into the blood, including their DNA and that can then be detected is really an amazing new technology, and it allows us potentially to understand properties of the cancer and follow them through blood tests rather through removing the tumor or biopsying the tumor. It isn't yet a complete replacement for tumor biopsies because there are things we can do with the tumor biopsy that we can't yet do with plasma. But we're increasingly learning much more of what we can do. It's an area that technology is developing quickly. I think research is going to enable us to use liquid biopsies to replace a lot of what we do with tumor biopsies in the future.
Dr. Sarat Chandarlapaty: It's a really important area of research because I think the upside of liquid biopsies is that it's relatively easy to collect things over time and as I mentioned, cancers evolve, cancers change, and we want to be able to track that. Moreover, the liquid may be sort of collecting from . if let's say a patient has a liver and a lung metastasis, well, the liquid, the blood is really sampling from both so we may be able to get information that's more comprehensive. So there are reasons why I think this is a very exciting technology, and I'm thankful that BCRF is helping to support research on it.
Chris Riback: Do you still remember what the reaction was? You presented that in San Antonio. It's, I think, about four and a half, five years ago, at this point. What was the reaction like for you around that work?
Dr. Sarat Chandarlapaty: Yes, I think people were very excited about the potential for this technology, and we just had another paper come out a month ago on following patients serially over time.
Chris Riback: Yes.
Dr. Sarat Chandarlapaty: On a clinical trial and seeing the evolution of the cancers through these liquid biopsies, through a blood test and just to know that we could use that to follow how the cancer was changing was really very powerful and we couldn't have done it otherwise.
Chris Riback: Yes, and the result of the most recent study was?
Dr. Sarat Chandarlapaty: That we saw these new mutations arise either in the estrogen receptor or in this other gene called P10. So this was a study where we were combining two drugs, an ER drug, and another drug, again, something called PI 3-kinase so a gene that's mutated in about 30 percent of breast cancers and this two-drug combination, which has been recently approved, we were finding that mutations were arising within a few months to either ER or PI3K and it told us that the cancer, if it could figure out even one of those two, that that might be sufficient to cause resistance and so we learned a lot about the timing of resistance and about the nature of it, what types of things were causing it.
Chris Riback: Yes.
Dr. Sarat Chandarlapaty: And so that's really informing us now about how to move ahead with a better sort of combination.
Chris Riback: Yes, I mean the liquid biopsy work can really guide treatment for women with metastatic breast cancer.
Dr. Sarat Chandarlapaty: Yes, I think that's right. It can enable us to know what mutations are present, which can guide our therapies, and can tell us when, why things aren't working when they aren't and perhaps more in the future.
Chris Riback: And where's the heart of your research right now?
Dr. Sarat Chandarlapaty : Yes, there's two kinds of I'd say big streams of research that are going on in my lab. First, we are trying to understand: What is the full sort of program? That is, estrogen talks to the estrogen receptor, the estrogen receptor talks the cell cycle, the cell cycle talks to the transcription program. Now I know that's a lot of terminology but there's a program, it's not just one gene. It's a whole pathway to change a cell from normal to cancer. What are all those steps, and in a resistant cell, wherein those steps did they become resistant? Because that tells you where you can attack with another drug, maybe a second drug.
So we're trying to understand the program better so that we can deal with resistance because I think if you can give two drugs in the program, it's very hard for the cancer to outsmart that. If you can give three new drugs in the program, it's almost impossible for the drug to outsmart that, and we've learned that with anti-microbial resistance, anti-bacterial resistance, that if you can really target things in a way that make it harder for the cell to evolve out of, then they don't come up with the solutions, yes.
The second is what I just said, there's this evolution that's happening. The cancers are developing new mutations. They're changing. There's this whack-a-mole sort of phenomenon, right? You hit the cancer with one drug, and then another tumor pops up and then you hit that one with another drug, and another pops up. Why? Because the cancer's evolving. It's changing and there's some basis for that. The other cells in our body aren't evolving, the cancer cells are evolving. How are they evolving, what's the process that's allowing them to change and adapt to our therapies? If we can figure that out, if we can develop anti-evolutionary sorts of medicines, then maybe we can just stick with the one drug and then block evolution.
Chris Riback: What's the hypothesis, what's the status of the anti-evolutionary drugs?
Dr. Sarat Chandarlapaty: We'll we're not at a drug stage yet, but we are increasingly understanding better, what are the sort of trajectories of cancers, how do they evolve. What we're doing is doing essentially a lot of human genome projects on cancer cells. We're doing lots and lot of DNA sequencing, not just once, but over time to say how did this cancer evolve, what were the changes? Then if you look back at those changes, you might interpret and understand what processes fueled them. So not in the realm of breast cancer commonly, but if you look at a lung cancer, you'll often see the imprints of smoking on the DNA. That is the types of mutations that smoking induces, leaves a signature. Similarly, if you look at melanomas, you will see an imprint or signature of UV sunlight damage and so we're looking for those kinds of imprints to tell us what kinds of things are changing. How is this cancer changing, compared to that one? Ultimately that’s leading us, I would say, to knowing the evolutionary process, and then we can go after it.
Chris Riback: And is the work you're describing, is this around the FoxA1 gene mutation? Is that the work that you're talking about right now or is that separate work?
Dr. Sarat Chandarlapaty: I'd say that's related. More of the work is on, for instance, this ESR1 mutation. Again, that's something that evolves. That doesn't happen at the beginning of breast cancer, that happens over time, typically with therapy. And another are these mutations in something like called FAT1, for instance, that's another one that seems to arise over time and the third one, I would say, is PTEN, that's another that we recently published on but these are all things that seem to be induced and not present necessarily at the very get-go.
Chris Riback: And is there any guidance or is there any practice, anything that you've seen where if the patients can do that can reduce risk of resistance or it's irrespective, that type of activity is just it's irrespective, just talk about another thing out of one's control. This is another that's out of one's control.
Dr. Sarat Chandarlapaty: Yes, this is not something that it's because we ate something or because we exposed ourselves to this that we see these things happen. These are intrinsic to the cancer and unfortunately, no, this is sort of out of control, but also I would say, not something one also should blame themselves for, so to speak and sometimes people do that. They'll blame themselves, "Oh, I shouldn't have done this or that," and that's not the case. This is unfortunately just the nature of these cancers.
Chris Riback: Yes, I think that's an important lesson for all folks to keep in mind. About you, how did you get into this and I mean going back, where did you grow up? I saw that you were educated, I think, in North Carolina at Wake Forest and then maybe another school in North Carolina as well, but was it always science for you? Was it always research even going back before university? Was this always where you knew you would end up?
Dr. Sarat Chandarlapaty: Yes, I'd always had interest in science, and my father's a physician. He did nuclear medicine when I was growing up in Miami, so I was exposed to that from the get-go. Then in college, I was really fascinated by chemistry and biology and so I actually pursued a PhD in biochemistry as my first stop. I didn't go to medical school. Then while I was in my graduate training, working on yeast cell biology, we were studying this pathway and at the time we were studying this pathway, it was also being found that that same pathway that was controlling yeast mating, was also being mutated in cancers. I was like, "Wow, that's pretty interesting. The same exact pathway, same set of proteins, and it plays a role in some cancers. I wish our understandings could inform that." I think that's when I realized I wanted to have a medical research sort of bent towards what my career end. Then I went to medical school and always with the intent of really doing sort of patient-centered research.
Chris Riback: And you know that insight that you just had that inspired you, the seeing an activity in one area of work and of life and applying it or making it, having it make you wonder about another part. I've got to say, one of the most interesting things that I've learned in these conversations is how leading researchers like you connect work across cancers and across different types of medicine. Do you find that . I mean, I understand that was at a different stage in your life and you were taking one area of research, it was taking you in one direction, and it opened up a whole other door for you. Part of your work today, requires you to be aware of and interact with different types of cancers. Is it the same thing? Items that you're learning about one area of cancer is that helping inform your work in breast cancer as well?
Dr. Sarat Chandarlapaty: Yes, absolutely. I mean, I think, we've really benefited so much by that sort of multi-disciplinary approach to science, and so there are countless examples. I mean one huge area in cancer has been the sort of understanding of immunology and then the potential for using, understanding immunology toward developing immune based cancer therapeutics. That's now become its own field. I think a lot of these are sort of these bridge fields that as you study very carefully, one area, cell biology and then you study another area, you realize that some of the findings in one area will inform the other. So it's what's exciting, and it truly brings innovation to what we're doing.
Chris Riback: What role has BCRF played in your research?
Dr. Sarat Chandarlapaty: Yes, so BCRF has been really essential in, I'd say two big ways I think about right now. One is just giving me a platform to explore new and innovative ideas. So if we have an idea and want to try something, BCRF recognizes that the only way we're going to develop really new technologies, new treatments is the spark of an idea, and so BCRF, by the way, it funds us. Obviously wants us to find really rigorous and good science, but it wants us to do things that are a little outside the box, too.
Dr. Sarat Chandarlapaty: And so as an example developing technologies to study cancer via blood test as opposed to via tumor biopsy, that was something that, you know, I didn't have a great deal of prior work on but we had an idea, and others had the technology and we worked with them and just having that funding from BCRF, to be able to explore that allowed us to find, for instance, the ESR1 mutation, was something that we widely see in blood tests and now blood tests are being used a lot for following cancers. But early on, many years ago, that was not something that was I could get a lot of funding for, so to speak. So I think innovation is one big area.
Dr. Sarat Chandarlapaty: And the second is just providing a network of investigators who can help each other out. So if I need, I'm studying a type of cancer, well, someone else might be developing models and that's what they do. They're developing all sorts of different models, and they're BCRF investigators, so they'll give me access to all their models and that's happened multiple times for me where I didn't have the specific type of mutation in the cancer models I had, but a BCRF person had and so we're all on the same team in trying to collaborate. That's been really a phenomenal resource for my lab and our work.
Chris Riback: Well, thank you, thank you for that collaboration.
Dr. Sarat Chandarlapaty: Yes.
Chris Riback: Thank you for the work that you do in your lab and every day.
Dr. Sarat Chandarlapaty: Thank you. Thanks for the chance to talk about all this.