The Daily Sign-Out

Is Notch signaling involved in lung carcinogenesis?

This is the question asked by Joyce Osanyingbemi-Obidi and colleagues from Johns Hopkins in an interesting recent paper published in Molecular Cancer Research.  The cancer stem cell hypothesis proposes that there is a small proportion of tumor cells with stem cell-like characteristics, including self-renewal capability, as well as a much larger proportion of "differentiated" cells with limited proliferative capability.  While stem cell-like cells have been characterized in a variety of malignancies, such a population for NSCLC has not been reported.  I previously blogged on a murine model for evidence of lung stem/progenitor cells. The authors cite the emerging body of data (perhaps not surprisingly) suggesting that the same key signaling pathways in embryonic and fetal development (e.g. Notch, Wnt, and Hedgehog) are also involved in the regulation of cancer stem cells.  The intracellular domain of Notch receptors (NICD) is released upon ligand binding and translocates to the nucleus where it converts the CSL complex from a transciptional repressor into an activator.  However, the NICD-CSL complex requires mastermind-like (MAML) proteins to recruit and bind coactivators that drive expression of downstream targets that promote cell growth and proliferation.  Aberrant Notch signaling has been described in a variety of malignancies (albeit with both activating and inactivating Notch mutations) and, indeed, elevated expression of Notch family members and a key downstream target, "hairy and enhancer of split 1" (Hes1), have been reported in NSCLC.

Using NSCLC cell lines showing overexpression of Notch3 and Hes1, the authors demonstrated that pharmacologic inhibition of Notch was associated with suppression of Hes1 expression and inhibition of clonogenic growth.  The authors used a clever LSL-KRAS^G12D mouse model in which an oncogenic KRAS allele is silenced by a transcription stop site flanked by lox P sites; inhalation of an adenovirus vector containing the Cre recombinase promotes rearrangment of the lox sites and deletion of the transcriptional stop and selective overexpression of mutant KRAS in bronchial and alveolar cells.  The authors showed a transient induction of Hes1 expression by immunohistochemistry in airway epithelial cells 1 week postinfection with adenovirus and gradually diminshing Hes1-positive cells in tumors over time.  They then used an novel inducible double-negative MAML knock-in murine model to show that inhibition of Notch signaling in vivo was not sufficient to suppress carcinogenesis or tumor progression in the context of mutant KRAS-driven carcinogenesis.

So--these data suggest that Notch signaling may have a role in early lung adenocarcinogenesis through the transformation of a tumorigenic stem cell-like cell but is not likely required for adenocarcinogenesis.  The authors provide several notable caveats and qualifications of this conclusion but, I think the key consideration is that Notch signaling is considerably more complex that we are given to understand.  Notch signaling in development can maintain stem cell characteristics in certain cell populations or drive cell differentiation in other.  Likewise, Notch pathway activation in neoplasia appears to have tumor-promoting and tumor-suppressing effects.  Also, not all Notch family members appear to equally contribute to lung neoplasia.  Finally, individual Notch isoforms may have opposing effects on carcinogenesis.

This paper may not be of general interest, especially for the busy practicing pathologist, but is worthwhile to reflect upon and ruminate over.  Intriguing stuff.

Hallmarks of cancer v. 2.0

In 2000, Douglas Hanahan and Robert Weinberg initially proposed six hallmarks of cancer that form an organizing principle for understanding the remarkable diversity of "cancer."  Their update, "Hallmarks of Cancer: The Next Generation," was published in the March 4, 2011 issue of Cell.

The six hallmarks of cancer are distinctive and complementary capabilities enabling tumor growth and metastatic dissemination and are a solid foundation for understanding the biology of cancer . These six hallmark capabilities of cancer, as originally defined, include the ability to sustain chronic proliferation, the ability to evade growth suppressors, the ability to resist cell death, the ability to achieve replicative immortalization, the ability to induce angiogenesis, and, finally, the ability to activate mechanisms for invasion and metastasis. These characteristics, common to all types of cancer, form a fundamental an understanding of the biology of cancer.

The authors use this review article, however, to update the massive progress that is been made in the last decade from their original article. They identify to enabling characteristics that allow cancer cells to acquire these functional capabilities. The most common is the development of genomic instability in cancer cells. This not only generates random mutations including chromosomal rearrangements but also rare genetic changes that can drive some of these hallmark capabilities.  A second enabling characteristic involves the inflammatory state of premalignant and frankly malignant lesions which serve to promote tumor progression through various mechanisms.

The authors highlight other distinct attributes of cancer cells which have been proposed as also being functionally important for the development of cancer and may also be added to the list of core hallmarks.  Two are especially compelling. The first involves the major reprogramming of cellular energy metabolism in order to support continuous cell growth and proliferation. The second involves active evasion by cancer cells from attack and elimination by immune cells. This latter capability highlights the dichotomous role of the immune system which can both antagonize and enhanced tumor development and progression.

The authors also include a very nice summary of the advances made in our understanding of the tumor microenvironment. They enumerate the various cell types that are known to contribute to the biology of the tumor microenvironment and also discuss some of the regulatory signaling that controls their individual and collective functions. Also included in their discussion is recent research showing the connections between the acquisition of cancer stem-like cells and the epithelial-mesenchymal transition differentiation program.

One interesting topic they discuss is the role of pericytes which is a cell type that I haven't thought of as being an important part of the tumor microenvironment. Pericytes represent a very specialized mesenchymal cell type that have fingerlike projections that wrap around the endothelial tubing of blood vessels. In normal cells or normal tissues, pericytes are known to provide paracrine support signals to the normally quiesent endothelium. The authors discuss research in which genetic and pharmacological disruption of the recruitment and association of pericytes which has demonstrated the functional importance of the cells in supporting the tumor endothelium. The authors conclude with a brief summary of possible therapeutic targeting of the hallmarks of cancer. They propose drugs that could interfere with each of the acquired capabilities necessary for tumor growth and progression and summarize some of which are involved in clinical trials or in some cases approved for clinical use in treating certain forms of cancer.

This is a long review article (hence the long post!) but well worth your effort and it is the most up-to-date and accessible review of our current understanding of the biology of cancer.

Use of oncology decision aids--by patients?!

Medscape reported on an article from J Clin Oncol that I thought was most curious:

September 1, 2010 —The use of decision aids by physicians caring for cancer patients is "low", say the authors of a Canadian study.

Only 24% of general surgeons, medical oncologists, and radiation oncologists in Ontario report using the aids, which are information guides that help patients make choices about cancer treatment.

The study, based on a survey of clinicians, was published in the May issue of the Journal of Clinical Oncology (2010;28:2286-2292).

However, in a letter published online August 30 in the journal, a German researcher raises an interesting possibility: that patients might be using decision aids at higher rates.

In other words, patients might be adapting this clinical tool more widely than the doctors who care for them.

I find this fascinating given the availability of such resources as Adjuvant! Online.  Research into the possible reasons for this discrepancy would be interesting.  Do doctors fear more than patients looking at "hard numbers" and probabilities?  or explaining or translating these numbers to patients in a forthright manner?  Or does the use of a decision aid somehow "dehumanize" a particular patient--the one sitting on front of you?  Your thoughts?

Mutations amok in lung cancer

A team from Genentech reports their findings in this week's Nature 465, 473-477 (27 May 2010) of direct tumor sequencing of a 51-year-old man with a 19-pack-year smoking history.  Note the greater than 50,000 mutations compared with paired "normal" lung. (egads!!!)  Not surprisingly, they found mutation of KRAS.  The magnitude and breadth of mutations is staggering and was surprising to the researchers.  Certainly give pause to ongoing efforts to identify oncogene-addicted tumors amenable to targeted therapy.

excerpt from abstract:

Here we present the complete sequences of a primary lung tumour (60× coverage) and adjacent normal tissue (46×). Comparing the two genomes, we identify a wide variety of somatic variations, including >50,000 high-confidence single nucleotide variants. We validated 530 somatic single nucleotide variants in this tumour, including one in the KRAS proto-oncogene and 391 others in coding regions, as well as 43 large-scale structural variations.

HealthDay has a nice accompanying article with an interview of the PI.

1-Cancer microenvironment: the role of stroma in neoplasia (more USCAP 2010)

This post is more from USCAP 2010.  I attended the "Advanced Molecular BIology" Special Course at USCAP on March 23.  One of the highlights was Dr. Rob West's presentation, "The Role of Stroma in Neoplasia."  I also had the pleasure of sharing a van ride with him to Dulles where we had a chance (because of miserable traffic) to talk for a long while about the cancer microenvironment, cancer stem cells, and, of course, our children.  What follows is my summary of his talk.

The role of the stroma in cancer can be summarized by two broad concepts:

• Wound response: "normal wound-healing gone awry"
• Cancer as an organ: altered communication between the cancer cells and the surrounding stroma

What are the components of the cancer microenvironment?

1. Myofibroblasts/fibroblasts
2. Endothelial cells (and bone marrow derived mesenchymal stem cells?)
3. Leukocytes, especially macrophages
4. Extracellular matrix

Myofibroblasts are key players in the cancer microenvironment because they modulate the extracellular matrix "landscape," regulate angiogenesis, and secrete growth factors and cytokines that influence a wide variety of cellular functions of cancer cells.

If the tumor is going to grow, the nascent tumor and surrounding stroma must recruit endothelial cells by expressing angiogenic factors (e.g. VEGF) which tips the balance of pro-angiogenic factors over anti-angiogenic factors.  Hypoxia, p53 mutation, decreased thrombospondin-1, increased HIF are other factors that promote angiogenesis and the angiogenic switch."  Furthermore, there appears to be an "endothelial-to-mesenchymal transition" (EnMT) that involves the development of vascular networks around the tumor composed of "tip cells"--vessel sprouts without vascular lumens.  This process is reminiscent of EnMT that occurs in tissue patterning during embryogenesis and is important for stabilizing the fragile neovasculature at the interface of the tumor and microenvironment.

The extracellular matrix is not a passive scaffold but is recognized now as an active, evolving compartment.  Invasion into the ECM by cancer cells is a multi-step process that involves dissociation and detachment from surrounding cancer cells, attachment to surrounding ECM, degradation of ECM, and migration through the remodeled ECM.  The other cellular components critically influence this entire process.

Macrophages are now recognized as pivotal in promoting tumor cell migration and invasion, angiogenesis, remodeling of the ECM, and the "metastatic niche."  In particular, macrophage subsets are being defined as "tumor-associated macrophages."

To be continued. . .

Cancer microenvironment--An interview with Dr. Robert Weinberg

The Wall Street Journal (subscription required) published an interesting interview with Dr. Robert Weinstein of the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts in which he discusses the role of the cancer microenvironment and its potential as a target for therapy.

Although directed toward a lay audience, this presents a well-structured overview and the accompanying podcast gives a little more depth.  It's well-worth a 7-minute time commitment.  I'm working on a near-future blog which will summarize a recent USCAP session I attended on cancer microenvironment and my discussion with the presenter.

The hazards of Hazard Ratios

In reading the cancer chemotherapy clinical trial literature, the unsuspecting yet intrepid pathologist finds himself amongst herds of hazard ratios.  I have tried to make sense of this for myself as follows.

"Time-to-event" curves plot the occurrence of an event over time for treatment and control groups and Cox proportional hazards regression is commonly used to analyze time-to-event curves.  The goal of a treatment may be to decrease the duration of the disease (like with antibiotic therapy) or to increase the duration of being "disease-free" (like with cancer chemotherapy) or alive (could be with or without disease).  The hazard ratio describes the relative risk of the event developing (such as disease recurrence in the case of cancer) for a treatment group compared to a control group over the course of time.  While hazard ratios have been commonly used to describe the extent to which a treatment can affect the duration of the interval of time to the event, this use can be misleading at best.  In other words, the hazard ratio is not an expression of the relative speed to an endpoint.  The HR may indicate a beneficial treatment effect and implies that the time to the endpoint was reduced/prolonged by the treatment but it provides no information on the magnitude of that time difference.  It is important to remember here that the proportional hazards model includes an assumption that the hazard ratio is constant over time.  What the hazard ratio does do is express the likelihood of the event occurring at a time-point in comparison between the treatment and control groups.  In other words, if the hazard ratio (HR) is 1 at time t, then there is an equal likelihood of the event occurring between the two groups (that is, no difference or effect); if the HR is 0.5, there is a 50% less likelihood of the event occurring between the two groups; and if the HR is 2.0, there is twice the chance or 100% more likelihood of the event occurring between the two groups.  It does not say that the patient has half or twice the speed to the event compared to the control.

If I'm interested in comparing the change in time to the event between two groups, then I need to use a time-based parameter, such as the median endpoint ratio.  Commonly, this literature reports the length of time from study entry to an endpoint for treatment and control groups and these are illustrated by a Kaplan-Meier curve.  Here one can derive a median time in which 50% of cases in each group have reached the endpoint and a mean time in which an average time to endpoint for each group.  Time-to-event analysis also has the advantage of including patients who fail to complete the trial or do not reach the study endpoint because one can compare the number of patients in each group at multiple points in time.  By looking at time-based parameters, I can thus get an idea of how fast each group reached an endpoint within a specified time frame.

I've heard this explained more simply as the HR telling you the chances of winning a race and time-based parameters telling you the margin of victory.

Phosphoproteomics reveals new ERK MAP kinase targets

Nature Structural & Molecular Biology 16, 1026 - 1035 (2009)
Published online: 20 September 2009

Although many extracellular signal–regulated kinase (ERK) mitogen-activated protein (MAP) kinase substrates have been identified, the wide diversity of ERK-mediated processes suggests the existence of additional targets. Kosako et al. report using a phosphoproteomic approach combining the steroid receptor fusion system, IMAC, 2D-DIGE and phosphomotif-specific antibodies, to detect 24 new candidate ERK targets showing phosphorylation changes between ERK-activated and ERK-inhibited samples.

I don't pretend to understand their experimental approach, but it seems novel and a brilliant way to analyze these events in a context-dependent manner.  This proteomics study is interesting because it is a functional study rather than a catalog of proteins identified in a static specimen.

They find that Nup50 is a legitimate ERK substrate and the authors find that ERK phosphorylation of the FG repeat region of Nup50 reduces its affinity for importin- family proteins, importin- and transportin. They also show other FG nucleoporins with a similar functional change after ERK-mediated phosphorylation. Moreover, they find that nuclear migration of importin- and transportin is impaired in ERK-activated, digitonin-permeabilized cells, as a result of ERK phosphorylation of Nup50. An explanation for the wide ranging effects of ERK phosphorylation may be that ERK phosphorylates various nucleoporins to regulate nucleocytoplasmic transport.

Also check out the accompanying "News and Views" commentary on the article by Natalie Ahn, "POREing over ERK substrates."

Leave well enough alone? The role of resection in asymptomatic metastatic CRC

The standard of care for treatment of mCRC has been surgical resection followed by adjuvant chemotherapy.  In patients presenting with symptomatic mCRC, palliative surgery is performed for bleeding, obstruction, or perforation, while in patients presenting with non-emergent symptoms and limited disease, surgery may be undertaken with a curative intent.  The role and value of surgery in patients with unresectable stage 4 colon cancer and a synchronous asymptomatic primary tumor is being currently investigated by the National Surgical Adjuvant Breast and Bowel Project in a prospective phase II study (NSABP C-10).  Current recommendations for treatment of mCRC are:

1. Patients with evidence of significant obstruction, perforation, or uncontrolled bleeding should undergo resection of the symptomatic lesion.
2. Patients who are unfit for surgery, or who decline surgery, should undergo endoscopic management with stents or ablation to palliate symptoms.
3. Patients with potentially resectable metastases should undergo resections of both the primary tumor and the metastases (typically, sequentially).
   1. Chemotherapy may be administered preoperatively to assess the natural history of the underlying malignancy.
   2. If disease controlled is obtained with chemotherapy, resection of primary and secondary lesions should be considered.
4. In patients who have diffuse, metastatic CRC with unresectable metastases, modern polychemotherapy with targeted agents will offer disease control without the mortality and morbidity of surgery directed at the primary lesion.

The hypothesis of this study is that, given the dramatic improvement in response rates with current regimens incorporating oxaliplatin and irinotecan with biologics such as bevacizumab and cetuximab, systemic chemotherapy without resection of the primary tumor is a reasonable alternative to the standard approach in these patients.  This approach takes into account the morbidity and mortality associated with colectomy in these patients compared to the potential survival benefit in an incurable disease where <10% of patients survive 5 years. 

Patients  will be treated with 5-fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) plus bevacizumab. The goal is to determine if this approach leads to an increase in “major morbidity” defined as any event related to the intact primary tumor necessitating surgery or resulting in patient death. The results of this study should be very interesting and could represent a real change in thinking about how we treat primary tumors. Oncologist. 2009;14(10):963-969.

Exosomes and nanoscale messenging

This month's Laboratory Investigation has an interesting free article by Hood et al. exploring exosomes--nanoparticles formed by inward budding of microvesiclar bodies and released into the extracellular matrix as part of a nanoscale messenger system.  The size of these particles (30-100nm) facilitates their penetration into cells at the invasive tumor front and they have been shown to participate in cell to cell communication such as morphogen and RNA transport between cells.

The authors' hypothesis is that melanoma exosomes act as "nanovehicles" (I love this term!) to prepare the microenvironment for tumor cell implantation.  This article describes a unique in vitro angiogenesis assay system to study exosomes and their influence on endothelial cell tubulogenesis (i.e., early anagiogenic changes) and spheroid sprouting (a later stage of angiogenesis).  This system also incorporates a novel method of tracking the trafficking of exosomes.

I know this is "out there" but I was not familiar with this topic at all and thought it fascinating.

P.S. Sorry for my recent prolonged absence from posting--teaching the second-year medical students some lung pathology.