Redirecting the Melanoma Tumor’s Microenvironment in Favor of the Activation of T-cells..Jim Breitfeller

Studies show that regulatory T (Treg) cells play a detrimental role in cancer immunotherapy because these cells accumulate in the tumor microenvironment and suppress immune responses. Moreover, Researchers recently showed the presence of tumor-specific CD4+, CD8+, and γδ Treg cells in several types of tumors, suggesting that they can induce antigen-specific, local immune tolerance at tumor sites. Tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MSC) could also play an important role in inhibiting immune responses and chronic inflammation, which has been linked to cancer development and progression. Both tumor-associated macrophages/DCs and MSCs promote tumor growth either by secreting immunosuppressive cytokines, including interleukin 10 (IL-10), transforming growth factor-β (TGF-β), and IL-1β, or by inducing Treg cell differentiation. More importantly, tumor cells have been shown to express inhibitory factors (IL-10, TGF-β, GAL-3, and IDO) to alter T-cell function. Immunosuppressive factors, such as FasL and TGF-β expressed by tumor cells, may directly inhibit tumor-reactive T-cell expansion or induce T-cell apoptosis. A recent studies suggest that tumor-associated galectin-1(Gal-1) and or Gal-3, a membes of the animal lectin family, contributes to tumor immune escapes by inhibiting the function of tumor-reactive T cells. Therefore, tumor cells constantly modulate T-cell responses by presenting tumor antigens and secreting immunoregulatory cytokines. Understanding the interplay between tumor cells and immune cells in the tumor microenvironment is essential for the development of effective cancer immunotherapy.

Researchers tested whether Gal-3 could activate other tumor-reactive or antigen-experienced T cells. Five melanoma-reactive T-cell lines, one prostate cancer–derived T-cell line, and two breast cancer–derived T-cell lines were selected and cocultured with 293 cells expressing Gal-3 for 12 to 16 h. they found that Gal-3–expressing 293 cells activated all of these T-cell lines to secrete IFN-γ but failed to activate naive CD4+ and CD8+ T cells purified from peripheral blood mononuclear cells (PBMC) of healthy donors . Suggesting that naive T-cell activation requires a strong T-cell receptor (TCR)-mediated activation, whereas tumor-reactive T cells can be readily activated by Gal-3. They also evaluated the cytokine profiles of CT28 T cells on Gal-3 stimulation. Gal-3 induced a high level of IFN-γ, granulocyte macrophage colony-stimulating factor, and low to middle levels of IL-4, which is similar to cytokine production induced by anti-CD3 (OKT3) stimulation. This is an indication that the Gal-3 complexes with the TCR synapse causing impaired signaling. Gal-3 binds and activates tumor-reactive T cells through carbohydrate-specific interaction.

Galectin-3 (GAL-3) localize mainly in Tumor cells, macrophages, epithelial cells,
Fibroblasts , and activated T-cells. Although galectin-1 has been shown to induce T-cell apoptosis, galectin-3 has conversely been shown to prevent cell death induced by Fas ligation. Galectin-3 has been shown to rescue cells from apoptosis by protecting against alterations of the mitochondrial membrane and formation of reactive oxygen species. A growing body of evidence supports the involvement of galectin-3 in tumor growth and metastasis.

Galectin-3 and IL-10 receptor needs to be inhibited to break the rest of the tolerance so the immunotherapy can have a greater effect with a better response rate.

Radiation + Yervoy + Anti-PD-1 + Anti-IL-10 receptor + GAL-3 Inhibitor= Robust immune response





“It is not the strongest of the species that survives, nor the most intelligent, but the one most responsive to change.”

~Charles Darwin~
Take Care,
Jimmy B

Déjà vu.. Blame it on the Tregs!! Melanoma..Jim Breitfeller

Déjà vu.. Blame it on the Tregs!!!!!!

As I reevaluate my theory, I have come full circle in my research and came across the research paper entitled:
The Role of Regulatory T Cells in Cancer
Tai-You Ha*
Department of Immunology, Chonbuk National University Medical School, Chonju, Chonbuk, Korea

The Role of Regulatory T Cells in Cancer






I postulate that the T regulatory cells may be the major stumbling block in the whole immunotherapy.
Recent studies have shown that CD4＋CD25high FoxP3＋ Treg cells are overrepresented in human metastatic lymph nodes with a 2-fold increased frequency
compared with both tumor-free lymph nodes and that advanced melanoma is associated with increased numbers of circulating Treg cells and Dendritic Cells (DCs) and suggested that melanoma induces immunosuppressive DCs and Treg cells in the systemic circulation of the patients . Vence et al also showed the presence of tumor antigen-specific CD4＋ Treg cells in the blood of patients with metastatic melanoma.

This means the host (you) may already have the correct antigen, but the tumor and microenvironment may be suppressing the immune response by secreting suppressive cytokines like TGF- beta , IL-10, and IL -6 and or proliferating the suppressive Tregs.

Nicholaou et al most recently showed in patients with melanoma that although strong antibody responses were mounted, the generation of delayed-type hypersensitivity response was significantly impaired and patients with advanced melanoma had a significantly higher proportion of circulating CD4＋ CD25＋FoxP3＋ Treg cells compared with those with minimal residual disease. So we now can blame the tolerance of our immune system to cancer on the T Regulatory Cells (Tregs).

It has been reported that the large number of different cell types that are claiming to be directly targeted by FoxP3＋ Treg cells are CD4＋, CD8＋ T cell, dendritic cells, B cells, marophages, osteoblasts, mast cells, NK cells, and NKT cells.


No wonder our immune system can’t raise an attack on the Melanoma. The tumor is like a castle in the middle ages. The castle (tumor) is surrounded by a moat the (suppressive cytokines) and the walls and towers are the Tregs. If you take out walls and tower, the castle becomes vulnerable.

These Tregs when activated, upregulate the CTLA-4, CD25 (IL-2), and other receptors. So if you control the function of the Tregs, you may be able to break the tolerance of the immune system. This can be done by blocking receptors on the Treg cells

It was also noted as the tumor burden increased, so did the Tregs in the peripheral which makes it harder to eradicate tumors in the advanced stage of Melanoma.



Wieczorek et al found that Treg cell numbers are significantly increased in the peripheral blood of patients with IL-2-treated melanoma. IL-2 stimulates CXCR4 expressed on the Tregs enables the Tregs to migrate CxCL12 in the tumor microenvironment increasing the Treg accumulation.

So we need to deplete, block and limit the expansion of the Tregs. We also want to limit the concentration of Interluekin-2 at the early stage of the CD4+ T-cell expansion. This can be done in a number of ways, but why not use an antibodies that can indirectly accomplish both tasks at once. By using anti-CTLA-4 blockage we keep the activation going, causing a free for all for the cytokine IL-2 that is secreted.

CD4+ T cells regulate immune responses by producing various cytokines upon antigen stimulation. Naive CD4+ T cells have limited cytokine responses and secrete only IL-2 before they differentiate into various effector cell types CD4+ T cells regulate immune responses by producing various cytokines upon antigen stimulation. Each cell type is competing for a limited amount of IL-2 secreted by a small subset of the T helper cells. Naive T cells stimulated with antigen in the presence of IL-4 differentiate into Th2 cells secreting IL-4, IL-5, IL-10, and IL-13, whereas IL-12 induces the differentiation of naive cells into Th1 cells secreting IL-2, IFN-γ, and lymphotoxin.



FROM:
Autoimmunity: IL-21: a new player in Th17-cell differentiation
Elissa K Deenick and Stuart G Tangye
WWW.Nature.com

Ribas and Restifo et al found that if you use Anti-CTLA-4 blockade the native T-cell is pushed towards the Th17 differentiation. This causes less interleukin-2 to be produced by the CD4+ T-cells causing a shortage of IL-2 in the microenvironment for the expanding T-cells thus limiting the differentiation to Tregs. It is well known that in the presence of TGF-beta and IL-2, the Native T-cell differentiates into T Regulatory Cells.

Also the activation and differentiation of Naïve CD8+ T cells require IL-2 provided by activated CD4+ T cells at the initial priming stage within 0–2.5 hours after stimulation. Thers is
critical IL-2 signal from CD4+ cells is mediated through the IL-2R (receptor) of Naïve CD8+ cells. This activation of IL-2 signaling advances the restriction point of the cell cycle, and thereby expedites the entry of antigen-stimulated Naïve CD8+ T-cell into the S phase of the cell cycle.

The cell cycle has four stages:

1. G1 phase when the cell increases in size and gets ready to replicate its DNA.
2. S phase when the cell synthesizes or copies its chromosomes
3. G2 phase in which the cell prepares to divide
4. M phase when mitosis occurs.

If no IL-2 is available to Naïve CD8+ T-cells, they never get a chance to expand and differentiate into Cytotoxic T Lymphocytes (CTLs) Killer T-cells.
Besides promoting cell proliferation, IL-2 stimulation increases the amount of IFNγ and granzyme B produced by CD8+ T cells.

Activation and Differentiation of (CTLs) Cytotoxic T Lymphocytes

Naïve CD8+ T-cells are referred to as CTL-precursors (CTL-Ps), which are incapable of performing any function other then recognizing the class I MHC-antigen complex on the Tumor cells through the (TCR) T Cell Receptor.

For activation, the CTL-P needs at least three signals:

1. Antigen specific signal transmitted by the MHC I peptide/TCR complex for the recognition of the Antigen

2. The Costimulatory signals transmitted by the CD28 receptor and the B7 molecule of the Antigen Presenting Cells (APCs)

3. Cytokine induced signal, IL-2 interaction with the IL-2r (receptor) on the CTL-P leading to activation and differentiation of the CTL-P into effector CTL

The Cytokine IL-2 came from the(CD4+) TH1 cells which means the CTL-P is IL-2 limited and can only be activated by the secreted IL-2 if there is any to be had or by introducing IL-2 through IL-2 therapy. Now you know the reasoning behind using the IL-2 therapy as the second part of the combinatorial Therapy. If there is not enough IL-2 in the host environment, you will only get partial expansion of the CTLs. It may not be enough to eradicate large bulky tumors.

The Interluekin-2 plays another role in this Melanoma Maze. In a study by Janas et al, Il-2 increases the expressions of the perforin and granzyme A, B and C genes in the CD8+ T-cells. This increase expression causes the CD8+ T-cells to mature into Cytoxic T Lymphocytes (CTLs). The exogenous IL-2 is required for the granzyme proteins. CTLs have cytoplasmic granules that contain the proteins perforin and granzymes. A dozen or more perforin molecules insert themselves into the plasma membrane of target cells forming a pore that enables granzymes to enter the cell. Once in the tumor cell, these enzymes are able to breakup (lyse) the cell and destroy it. This is the beginning of the end for the cancer cells. The tumors begin to shrink and the rest is history.

Research suggests that the primary mode of the destruction of the tumors by CTL is by initiating death through the Fas-FasL pathway. Studies have shown that CTLs store Cytotoxic proteins in the form of granules in their cytoplasm.

These proteins belong to two categories:

1. Perforins: involved in pore formation
2. Granzymes: responsible for hydrolysis of the cellular products.

Granzymes breaks down the tumors cells just like your detergent enzymes in your laundry detergent.

Immediately following a CTL contact with the tumor cell, the Golgi sacks load with granules and granzymes which create pores to allow the granzymes to enter and destroy the tumors cells.

So now you know why Anti-CTLA-4 blockage and HD Interluekin-2 go hand and hand. Timing of the addition of IL-2 can make or break the immune response. And without suppressing the Tregs, your chances to mount an immune response may be slim at best. Your Immune System is a well Orchestrated system of events; we just need the Knowledge on how to harvest its potential on eradicating cancer.

The beginning of knowledge is the discovery of something we do not understand.
~Frank Hebert~
A critically acclaimed and commercially successful American science fiction author


Take Care,

Jimmy B

“CD4+ T-regulatory cells: toward therapy for human diseases”Melanoma ..Jim Breitfeller

“CD4+ T-regulatory cells: toward therapy for human diseases”

Thanks to Dr. Megan Levings for taking time out of her busy schedule to email me her research paper on Tregs cells. In my research, I have come to the conclusion that that major barrier to initiating an immune response is over coming the Treg suppression. Remember I said “BLAME IT ON THE TREGS”, well most of the research finding are supporting this piece of the Melanoma Puzzle.

“T-regulatory cells (Tregs) have a fundamental role in the establishment and maintenance of peripheral tolerance. There is now compelling evidence that deficits in the numbers and/or function of different types of Tregs can lead to autoimmunity, allergy, and graft
rejection, whereas an over-abundance of Tregs can inhibit anti-tumor and anti-pathogen immunity.”

“Several different types of Tregs exist in humans, including specialized subsets of CD4+, CD8+, double negative CD3+ CD4-CD8-, alpha /gamma T cells, and natural killer T (NKT) cells. While it is likely that these different types of Tregs work together in a network to maintain immune homeostasis, the majority of current research is focused on defining the normal function of CD4+ Tregs, because these cells mediate dominant, long-lasting, and transferable tolerance in experimental models.”

Levings and colleagues want to harness the Tregs to develop adoptive cellular therapy protocols that would be used in autoimmunity and inflammation. So they wanted to grow the population of Tregs. We on the other hand want to deplete/suppress them, to push the balance towards an immune response.

The Tregs constitute only 1-2 % of the peripheral CD4+ T cells in humans, but is enough to keep the immune system in check. In in-vitro culture conditions, they can expand rapidly when stimulated with an antigen. If you throw Il-2 into the mix, It can increase the cell population by 5-6 fold. This means that the Treg population when activated and Il-2 is secreted or added like (HD IL-2), is now at 10 to 12 % of the population. This can quickly shut down the immune response. So the Takeaway is that one must plan when to introduce the Il-2 to the therapy. So Lets follow nature. Il-2 Is secreted after the CD4+ T cells are activated. THIS MEANS TIMING OF THE IL-2 IS CRUCIAL TO GENERATING A RESPONSE. How Long do we want to hold off before inoculation of the IL-2? We need the growth profiles of each of the CD4+ T cells.

Based on the data gather by Itoh and Colleagues we want to add the Il-2 at the Maximum propagation of the CD8+ T cells. I t has been reported that when the CD8+ T-cells are grown in the presence of Il-2 , they develop in to (CTL) Cytotoxic T Lymphocytes. That is just what we wanted.

This is where Itoh and Colleagues come in:

In 1988, a paper was published “Autologous Tumor Specific Cytotoxic Lymphocytes in the Infiltrate of Human Metastatic Melanomas Activation by Interleukin 2 and Autologous Tumor Cells, and Involvement of the T Cell Receptor “ by Itoh and Colleagues. In their studies, they propagated (TILs) Tumor infiltrate lymphocytes cells from 12 Metastatic Melanoma patients. They preformed kinetic growth studies in IL-2 and even broke it down three Surface markers (CD3, CD4 and CD8).

The results are as follows:
The Total average maximum propagation was 43 days. (N=12)
The average maximum propagation for (lung, Axilla) was 40 days (n=3)

The average maximum propagation for (CD3) was 78 +/- 11 days (n=12)

The average maximum propagation for (CD4) was 33 +/- 10 days (n=12)
The average maximum propagation for CD4 (lung, Axilla) was 26 days (n=3)

The average maximum propagation for (CD8) was 49 +/- 17 days (n=12)
The average maximum propagation for CD8 (lung, Axilla) was 57 days (n=3)

Base on the above data, it would take about 49 days for my activated CD8+ T cells to reach maximum propagation. Then add the IL-2.

The Orchestration of an Inmmune Response Unrehearsed

Source: All Quotes are excerpts from Dr. Megan Levings’s Paper

So now if you put a clinical trial together and follow the timelines,

So now lets see My actual timeline.


They Always said Timing is Everything!!!!!


Take Care,

Jimmy B

Investigators have seen increased Treg cells in lymph nodes with Melanoma! Jim Breitfeller

"Other investigators have demonstrated an increased proportion of Treg cells in lymph nodes with metastatic melanoma [28]. Furthermore, the failure of an objective clinical response in patients undergoing immune therapies has been mechanistically linked with elevated percentages of Treg cells in these patients [29, 30]. Although the percentage of Treg cells was relatively small in our study (ranging from 1.3% to 3.7% of CD4+ T cells), Treg cells are quite active in small concentrations and have been shown to inhibit cellular proliferation and cytokine secretion in effector cells in in vitro studies at concentrations as low at 3% of CD4+ T cells [31]. In addition, the immunosuppressive activity of Treg cells follows a dose response curve and small increases in their concentration greatly enhance their immune suppression [31]. These findings, taken together with the results of our study, point toward a mechanism of immunosuppression in melanoma involving Treg cells."

source:http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2043471

Treg cells in lymph nodes with metastatic melanoma



I know this paper is very technical, but it is the needle in the hay stack. If we can contol the Tregs, than we have a fighting chance of involking an immune repsonse.

Please take a moment to read this paper.

Take care

Jimmy B

Tumors Evading Detection!!!!! Melanoma.. Jim Breitfeller

Tumors Evading Detection
Lack of costimulation:

Many Melanoma tumor cells do not have the B7 protein on their surface so this co-stimulatory second signal cannot take place. Theoretically, they should cause an immune response but they do not stimulate an effective anti-tumor immune response. The first signal originates from the binding of the T cell receptor (TCR) to its antigen-MHC, and provides the specificity of the interaction. Without this signal, the cell enters anergic state and can act as a T reg cell. Expression of B7 on the surface of a cell is the costimulatory signal necessary to allow for the cytolytic CD8+ T cell attack on the tumor. B7 display renders tumor cells capable of effective antigen presentation, leading to their eventual eradication.

Secretion of immunosuppressive cytokines:

Another way tumors evade detection is by secretion of certain cytokines. They are low-molecular weight proteins that use their communication ability to regulate the immune response. Cytokines can act upon either the cells secreting them (autocrine) or on neighboring cells (paracrine) to generate activities in the targeted cells. This means they can act as light switches for on and off immune responses. For example, interleukin-2 activates a cell-mediated immune response, while interleukin-10 suppresses cell-mediated responses. Many types of cancer, including Melanoma, take advantage of this ability to down regulate this appropriate immune response to help extend their survival and proliferation. This causes cancer patients to fail in mounting a successful attack on the tumors. Immunosuppressive cytokines secreted by cancer cells include transforming growth factor-beta (TGF-beta), interleukin-10 (IL-10) and vascular endothelial growth factor (VEGF).

TGF-beta is one of the most potent immunosuppressive cytokines characterized to date. It is capable of affecting the proliferation, activation and differentiation of cells participating in both the innate and acquired immune response.TGF-beta inhibits the profilation T-cells, B cells, Natural killer cells (NK), and macrophages.TGF-beta also converts T-cells, which normally attack cancer with an inflammatory (immune) reaction, into regulatory (suppressor) T-cells, which turn off the inflammatory reaction. Another of TGF-beta's affect is on cytotoxic T lymphocytes (CTLs) This is very important for anti-tumor immunity because of their cytotoxic effects. TGF-beta down-regulates many of the processes necessary for CTL activation. Without this activation, there is no assault on the tumor cells from the CTLs. In addition to suppressing proliferation, TGF-beta has been shown to induce apoptosis (cell death) in B and T cells.

Another immunosuppressive cytokine is IL-10. It is capable of inhibiting the prodction of of pro-inflammatory cytokines like IFN-gamma, IL-2, and GM-CSF made by cells such as macrophages and T helper cells. IL-10 also displays potent abilities to suppress the antigen presentation capacity of antigen presenting cells. Secretion of IL-10 in the vicinity of a tumor can render the tumor totally insensitive to CTL-mediated lysis. It is most likely that the tumor’s microenvironment is altered enough to block or turn off the discharge granules that would lyses the tumor cell. However, it is also stimulatory towards certain T cells, mast cells and B cells. It enhances B cell survival, proliferation, and antibody production. As you can see, IL-10 has many rolls to play when it come to the immune system.

The cytokine VEGF is produced by most tumors.
Vascular endothelial growth factor (VEGF) is a cytokine that is produced by most tumors. This growth factor enables the tumor to expand vascularly when is in its growth phase. VEGF production can be induced in tumor cells that are not receiving enough oxygen.

Regulatory T-cells (Tregs) (suppressor T cells) are a specialized subpopulation of T cells that act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens.1

Tumor Growth kinetics

The cell cycle has four stages:

1. G1 phase when the cell increases in size and gets ready to replicate its DNA.
2. S phase when the cell synthesizes or copies its chromosomes
3. G2 phase in which the cell prepares to divide
4. M phase when mitosis occurs.

When the various growth inhibitory proteins and checkpoint controls which regulate this cycle become disabled due to mutations characteristic of cancerous cells, the cell cycle is no longer under tight regulation. Tumor cells are capable of proliferating so quickly that the immune response is not fast enough to keep their growth in check. The growth of the tumor cells outpaces the immune response and escape the detection of the immune system. Lack of cell cycle controls leads to excessive proliferation of tumor cells.





I am getting closer!!!!

Take care

Jimmy B

IL-2 Regulates Perforin and Granzyme Gene Expression in CD8+ T Cells Independently ..Melanoma.. Jim Breitfeller

IL-2 Regulates Perforin and Granzyme Gene Expression in CD8+ T Cells Independently of Its Effects on Survival and Proliferation

I know this doesn't sound exciting to you, but it does to me. See I am slowly piecing the puzzle together that made my Sequential Cancer Treatment work so far.If I knew how complex the biochemistry of our immune system was, I would asked for cliff notes.


"Granule-mediated cytotoxicity is one of the major mechanisms used by CD8+ T cells to eliminate harmful or foreign bodies, such as virus-infected cells, tumors, and allografts. After Ag recognition, activated CD8+ T cells release the contents of their cytotoxic granules into the extracellular space, where they are taken up by the target cell, and apoptosis is initiated (1). The cytotoxic granules contain a number of molecules, including the pore-forming protein, perforin, and serine proteases, known as granzymes. Perforin was originally thought to cause cell lysis by penetrating the target cell membrane (2), but recent work favors the theory that perforin functions by enabling the granzymes to escape from endosomes into the cytosol of the target cell (3, 4). Whatever its exact role, perforin is essential, because Ag-specific granule-mediated cytotoxicity is absent in perforin-deficient CD8+ T cells and NK cells (5)."

I think I can do some hand waving but I still need to know how my immune system was able to differentiate between self (the body) and non-self (the tumor). I believe one of the keys may be the T-reg cells.

"These studies suggest that IL-2 itself may either directly or indirectly play an important role in the development and/or function of this unique population of CD25+ suppressor cells."

Source:http://jem.rupress.org/cgi/content/full/188/2/287

CD4+CD25+ Immunoregulatory T Cells Suppress Polyclonal T Cell Activation In Vitro by Inhibiting Interleukin 2 Production By Angela M. Thornton and Ethan M. Shevach



"Regulatory T cells (sometimes known as suppressor T cells) are a specialized subpopulation of T cells that act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens. The existence of a dedicated population of suppressive T cells was the subject of significant controversy among immunologists for many years. However, recent advances in the molecular characterization of this cell population have firmly established their existence and their critical role in the vertebrate immune system. Interest in regulatory T cells has been heightened by evidence from experimental mouse models demonstrating that the immunosuppressive potential of these cells can be harnessed therapeutically to treat autoimmune diseases and facilitate transplantation tolerance or specifically eliminated to potentiate cancer immunotherapy.

T regulatory cell populations

T regulatory cells are a component of the immune system that suppress immune responses of other cells. This is an important "self-check" built into the immune system so that responses do not go haywire. Regulatory T cells come in many forms, including those that express the CD8 transmembrane glycoprotein (CD8+ T cells), those that express CD4, CD25 and Foxp3 (CD4+CD25+ regulatory T cells or "Tregs") and other T cell types that have suppressive function. These cells are involved in closing down immune responses after they have successfully tackled invading organisms, and also in keeping in check immune responses that may potentially attack one's own tissues (autoimmunity).

CD4+Foxp3+ regulatory T cells have been referred to as "naturally-occurring" regulatory T cells to distinguish them from "suppressor" T cell populations that are generated in vitro. The regulatory T cell field is further complicated by reports of additional suppressive T cell populations, including Tr1, CD8+CD28-, and Qa-1 restricted T cells. However the contribution of these populations to self-tolerance and immune homeostasis is less well defined."

Source:Wikipedia

Back to the books

Oncogene Inhibits Tumor Suppressor To Promote Cancer: Study Links B-RAF And LKB1..Melanoma .. Jim Breitfeller

Oncogene Inhibits Tumor Suppressor To Promote Cancer: Study Links B-RAF And LKB1

Scientists have uncovered an interesting connection between two important protein kinase signaling pathways that are associated with cancer. The research, published by Cell Press in the January 30th issue of the journal Molecular Cell, may direct new therapeutic strategies for multiple types of cancer.


The protein kinase LKB1 is a known tumor suppressor and the LKB1-AMPK signaling pathway couples energy metabolism with cell growth, proliferation and survival. “Mutations in LKB1 are not frequent in human cancers and it is not clear how tumor cells suppress the signaling pathway to gain growth advantage under conditions of energy stress (common in cancer cells),” explains senior study author Dr. Lewis C. Cantley from Beth Israel Deaconess Medical Center and Harvard Medical School.

Dr. Cantley and colleagues, including Dr. Bin Zheng, designed a study to investigate the molecular mechanisms associated with suppression of the LKB1-AMPK pathway in tumor cells. The researchers used malignant melanoma cells that often have a mutation called “V600E” in the RAF protein B-RAF. The RAF-MEK-ERK pathway is well established as a key regulator of cell growth, proliferation, differentiation and survival.

Mutations in the RAF kinase B-RAF have been found in many types of human cancer but, while oncogenic B-RAF V600E has been linked with tumor induction, growth, maintenance and progression, the specific molecular mechanisms have not been identified. Dr. Cantley’s group found that melanoma cells with the B-RAF V600E mutation had impaired AMPK activation and that inhibition of B-RAF signaling activated AMPK.

The researchers went on to show that LKB1 was phosphorylated by two kinases that are downstream of B-RAF, ERK and Rsk. The phosphorylation of LKB1 interfered with the ability of LKB1 to bind and activate AMPK. Importantly, expression of mutant LKB1 that could not be phosphorylated resulted in activation of AMPK and an inhibition of melanoma cell proliferation.

“Taken together, our results provide a molecular linkage between the LKLB1-AMPK and the RAF-MEK-ERK pathways and suggest that suppression of LKB1 function by B-RAF V600E plays an important role in B-RAF V600E-driven tumorigenesis,” says Dr. Zheng. “It’s conceivable that tumor cells must turn off the LKB1-AMPK signaling pathway to gain a growth advantage under conditions of energy stress.”

Given that B-RAF mutation and loss of LKB1 are associated with multiple types of cancer, the work is likely to have a significant clinical impact. “Further understanding of how the intriguing molecular linkage between LKB1-AMPK and RAF-MEK-ERK functions in tumorigenesis could potentially provide great therapeutic opportunities for cancer treatment,” offers Dr. Cantley.

The researchers include Bin Zheng, Harvard Medical School, Boston, MA; Joseph H. Jeong, Dana-Farber Cancer Institute, Boston, MA; John M. Asara, Harvard Medical School, Boston, MA; Yuan-Ying Yuan, Harvard Medical School, Boston, MA; Scott R. Granter, Brigham and Women’s Hospital, Boston, MA; Lynda Chin, Dana-Farber Cancer Institute; and Lewis C. Cantley, Harvard Medical School, Boston, MA.

Source: Cathleen Genova
Cell Press

Jimmy B