The "PAK" Story

Leading to a New Therapy of Cancers and NF

The origin of PAK

The new word "PAK" was created by a Brit scientist called Edward Manser
around 1994. Ed and his colleagues at IMCB of Singapore National University then cloned
a new gene from mammalian brain, which encodes a new enzyme, Ser/Thr kinase,
that is uniquely activated by two GTPases (or G proteins) called Rac and
CDC42 of 21K daltons. Then these small GTPases were collectively called
p21. So he proudly coined this new enzyme "PAK" (standing for p21-activated
kinase). Unfortunately, however, just a year before, another protein of
21K daltons was cloned, and named p21, WAF1 or CIP1, depending on its discoverers.
This new protein (p21/WAF1/CIP1) has nothing to do with these small GTPases,
but an inhibitor of another kinase called CDK (cyclin-dependent kinase).
To avoid the potential confusion, many "wise" experts in this kinase field
decided not to use the terminology p21 for a very large family of small
GTPases any more, and began to use only the simple word "PAK" for this Rac/CDC42
(p21)-dependent Ser/Thr kinase. On the other hands, this CDK inhibitor of
21K daltons continued to be called "p21" world wide till now.

Since then so many members of PAK family kinase have been cloned from mammalian
tissues, and called PAK1, PAK2, PAK3, PAK4, PAK5, PAK6 and so forth. Also
another family of Rac/CDC42-dependent Ser/Thr kinases was discovered in
mammals, and called "MLK" (standing for mixed linage kinase). Well, back
to 1977, when I used to work at Ed Korn's lab of NIH, we discovered a new
Ser/Thr kinase in a soil amoeba, which phosphorylates the heavy chain of
a unique single-headed myosin called Myosin I. So we called this kinase "Myosin
I heavy chain kinase" (MIHCK). This kinase is essential for the activation
of this myosin ATPase by F-actin (actin filaments) . Shortly after PAK1
was cloned from mammals, we compared the amino acid sequence of PAK1 and
MIHCK, and found that these two kinases are very closely related. Both enzymes
contain the Rac/CDC42-binding site at N-terminal half, and the kinase (catalytic)
domain at C-terminal half. Furthermore, like PAK1, MIHCK is indeed activated
by Rac/CDC42. In other words, our MIHCK was the first identified member
of PAK kinase family, perhaps called "PAK0" (O for "zero" or "origin"). Yes,
both MIHCK (PAK0) and PAK1-3 (class I PAK) are involved in the regulation
of non-muscle cell movements such as membrane ruffling and cell migration
in primitive amoeba and mammalian cells.


PAK-dependent diseases

During last decade, PAK (in particular PAK1) research entered medical field,
in particular molecular oncology. The major reason was that the GTPases
Rac and CDC42 were found by Frank McCormick's group of Onyx Pharm during
1995-1996 to be essential for RAS-induced malignant transformation of normal
fibroblasts such as NIH/3T3 cells. RAS, the small GTPase of 21K daltons,
is mutated oncogenically in more than 30% of all human cancers. Most notably,
RAS is mutated in more than 90% of pancreatic cancers, and more than 50%
of colon cancers. Oncogenic RAS somehow activates both Rac and CDC42 to
cause these cancers. In 1997, Jeff Field's group at University of Pennsylvania
found that PAK1 is essential for RAS-induced malignant transformation of
a fibroblast. This discovery triggered many scientists including us to investigate
further the potential role of PAK1 in many other cancers and even several
other diseases.

In 1998, Jeff's group reported that PAK1 is essential for the growth of
NF1 tumor in mice. NF stands for neurofibromatosis, a group of genetic diseases often associated
with tumors in brain, along spine and on skin. There are two types of NF.
Type 1 (NF1) is caused by dysfunction of NF1 gene product, a RAS GAP of
2818 amino acids. (Loss-of-function) mutation of this tumor suppressor gene
causes the abnormal activation of RAS, which is very similar to oncogenic
mutation of RAS, and eventually leads to the abnormal activation of PAK1.
Type 2 (NF2) is caused by dysfunction of NF2 gene product called "Merlin"
which we recently found is an inhibitor of PAK1, another tumor suppressor.
Thus, dysfunction of "Merlin" also causes the abnormal activation of PAK1.
NF2 is associated with brain/spine tumors called Meningioma and Schwannoma.
In other words, RAS cancers such as pancreatic and colon cancers are closely
related to both NF1 and NF2 tumors at molecular levels, being caused by
the oncogenic PAK1. However, until recently no effective therapeutics was
available on the market for these cancer or NF patients.


Anti-PAK drugs

Thus, during last several years, we have identified or developed a series
of anti-PAK1 drugs from synthetic chemicals and natural products for the
therapy of these PAK1-dependent cancers/tumors. Among them, so far the antibiotic
(a ring peptide) called FK228 is the most potent. It was originally developed
by Fujisawa Pharm around 1994 for suppressing the growth of RAS cancer cells,
and then later found to be a specific inhibitor of HDAC (histone deacetylase)
which we found eventually blocks the oncogenic PAK1 pathways. FK228 is now
in clinical trials (phase 2) mainly for CTCL (cutaneous T-cell lymphoma).
However, in animal models, FK228 was found to suppress the growth of so
many other cancers such as breast, prostate, pancreatic cancers, NF tumors,
and glioma, which altogether represent more than 70% of all human cancers.
Furthermore, FK228 suppresses several other diseases such as asthma and
arthritis in mouse models. These findings strongly suggest that PAK1 is involved
in the majority of cancers, and even several non-tumor diseases. Using other
anti-PAK1 drugs, we and others confirmed that formidable blood cancers such
as MM (multiple myeloma), and Alzheimer diseases (AD), Parkinson's diseases
(PD), AIDS (HIV-infection), epilepsy, malaria and autism such as Fragile
X syndrome also require PAK1. These additional findings hint the potentially
huge market value of these anti-PAK1 drugs. However, among these synthetic
anti-PAK1 chemicals, so far only one drug is available on the market with
FDA approval. It was originally called "SAHA", but Merck brand-named "Zolinga"
in October of 2006 only for the treatment of CTCL. Like FK228, SAHA is
an HDAC inhibitor, but less potent than FK228.


Anti-PAK drugs in Nature

Since clinical trials of these anti-cancer/anti-PAK1 drugs usually would
take a decade or more to be completed for each drug to get ready for marketing,
none of these cancer or NF patients would get any immediate benefit from
these potent anti-PAK1 drugs such as FK228, CEP-1347, TAT-PAK18, OSU-03012,
and GL-2003/GL-2005 for the coming decade. Thus, a few years ago, responding
to the urgent cry from many cancer/NF patients or their families, we decided
to identify an effective anti-PAK1 product in the nature, that is among all
sorts of vegetables, fruits or healthcare food supplements available on the
market.

After try and error (and educated guess), we eventually found the first effective
natural anti-PAK1 product. It has been sold as a healthcare food supplement under
the brand name of "Bio 30" mainly in New Zealand (NZ). Interestingly, the million-
years wisdom of NZ honey bees has contributed to this recent finding of ours.
Bio 30 is produced by bee keepers outskirts of Auckland where Sir Edmond
Hillary (1919-2008) used to live. He was best known to reach first the summit
of Mt. Everest (8850 m high) with his SherpaTenzing Norgay in 1953, but
also known as a life-long bee keeper. It has been said for many decades
that no bee keeper suffers from any cancer. Sir Edmond indeed was never
caught by cancer. However, nobody knew why.

Two decades ago, however, a Jewish scientist at Columbia University found
the first clue to this mystery. Dr. Grunberger identified the major anti-cancer
ingredient in propolis extract. It was CAPE (caffeic acid phenethyl ester).
Honey bees collect CAPE mainly from young buds of poplar trees, making "honey
wax" (also called "propolis" first by Hippocrates) to seal the honeycombs,
protecting their larva from all sorts of pathogenic bacteria or viruses.
Because of its anti-infectious action, ancient Egyptian people used propolis
for healing wounds and preparing mummies. Interestingly, the CAPE content
of NZ propolis turns out to be the highest among propolis samples around
the world. Indeed around Auckland there are so many poplar trees.

CAPE down-regulates the GTPase Rac, and eventually inactivates PAK1. During
2006-2007 we found that Bio 30, CAPE-rich water-miscible extract of NZ propolis,
can suppress the growth of RAS cancers such as pancreatic cancers, NF tumors,
and breast cancers in mice most effectively. Thus, many patients, in particular
NF and pancreatic cancer patients, began to take Bio 30. Bio 30 costs only
a dollar for daily treatment, being among the least expensive anti-cancer
drugs, and is very safe. The only minor problem with Bio 30 is that 1-2
% of people turns out to be allergic to CAPE in this propolis extract, developing
an itchy rash on their skin.

http://www.ncbi.nlm.nih.gov/pubmed/18726924?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

For those who are allergic to CAPE, Brazilian green propolis extract (GPE)
would do the same job, suppressing the growth of NF tumors and RAS cancers,
and does not cause any allergy, although GPE is much more expensive than
Bio 30 on the market. The major anti-cancer ingredient in GPE is ARC (Artepillin
C), and we recently confirmed that ARC also selectively blocks the oncogenic
PAK1 pathway. This year a report said that red propolis extract (RPE) from
Brazil also suppresses the growth of PAK1-dependent pancreatic cancer cells,
suggesting that RPE also blocks PAK1 pathway, although RPE contains neither
CAPE nor ARC. In other words, honey bees wherever they live must have developed
a unique instinct (or biological mechanism) to detect that CAPE or ARC or
a third anti-cancer ingredient in RPE are anti-PAK1, through their million-years
experience.


An addiction for healthier and longer life

In a tiny nematode of just 1 mm long called C. elegans, we recently discovered
that PAK1 increases their heat-sensitivity and shortens their life span
by inactivating a tumor suppressor called Foxo. When this worm is treated
with anti-PAK1 drugs such as CAPE and ARC (or PAK1 gene is knocked out),
they become very heat-resistant (acquire thermo-resistance), lay far fewer
eggs and have a prolonged life span. Thus, it is likely that honey bees
also feel quite comfortable or even happy somehow when they take-in sugars
(glucose or fructose) or anti-PAK1 ingredients such as CAPE and ARC from
their favorite flowers or trees, and they have developed a sugar/anti-PAK1
addiction over centuries. Such an addiction would help honey bees collect
sugar for making "honey", and anti-PAK1 ingredients for making "propolis"
to feed and protect their larva in honeycombs.

Interestingly, we, human beings, also carry a similar Foxo gene(s) that
is responsible for suppressing tumors and prolonging life span. So if people
also develop such an anti-PAK1 drug addiction, they could survive the escalating
global warming and enjoy a cancer /disease-free long life. The optimum temperature
for normal (wild-type) nematodes is around 20oC, and they would die at 35oC
in a few hours. However, if PAK1 gene is deleted, they would live happily
even at 35oC. Recently the major anti-cancer ingredient in red grape called
resveratrol (or trans R3) can activate Foxo, leading to a longer life span,
by both inactivating PAK1 and activating another kinase called AMPK which
is responsible for the regulation of glucose up-take. Thus, the R3-rich
red wine/grape juice would be good not only for treating diabetes and obesity,
but also for leading a cancer-free healthier life.

In short, the historic discovery of mammalian PAK by Ed Manser's group opened
a new "natural" avenue/approach leading us to the much more improved quality
of life, over a decade detailed basic research by my own group and many
others.