Chemosenstization of Inflammatory Breast Cancer by Curcumin

Background: Inflammatory breast cancer (IBC) is a rare (2-6% of all breast cancer) but very aggressive, rapid,
and most lethal form of breast cancer affecting mostly younger women and extremely rarely older men. The
three-year survival rate for patients with IBC is 40%, which is significantly lower compared to near 90% survival
rate for patients with all types of breast cancer combined [1, 2]. What makes IBC different from other types of
breast cancer, is that symptoms progress very rapidly often within matter of weeks, exhibits aggressive
histological features, exhibits high incidence of local and systemic recurrence, manifests an exaggerated
degree of lymphovascular invasion in situ and is inversely associated with socioeconomic position [3]. At the
time of diagnosis, 30% of the IBC patients have distant metastasis as compared to 5% of the non IBC patients
[1]. Although paclitaxel is used as therapeutic for IBC patients, the drug alone is unsafe and ineffective [4].
Thus, agents that can enhance the effects of paclitaxel and/or overcome patients’ resistance to the drug are
needed for the treatment of IBC.
At molecular level IBC generally exhibits high S phase, aneuploidy, negative for estrogen and progesterone
receptors, p53 mutations, increased expression of EGFR (30% of the pts) and HER2 amplification [5-8]. In
addition overexpression of an oncogenic RhoC GTPase which controls the angiogenic factors has been linked
to IBC [9]. Overexpression in gene products MUC1 and calcium-dependent adhesion molecule E-cadherin
have also been linked to human IBC tumors. Moreover loss of tumor suppressor LIBC/WISP3 is associated
with the pathogenesis of IBC.
The feature that distinguishes IBC from other forms of breast cancer, are rapidly progressive breast
inflammation and an extreme tendency to metastasize; both activated by proinflammatory transcription factors
NF-B and STAT3 [9, 10]. NF-kB activation has been linked with transformation, survival, proliferation,
invasion, angiogenesis, and metastasis [11, 12]. In fact, microarray-based studies, genome-wide expression
profiling studies, NF-kB DNA binding assays, and real-time RT-PCR studies; all have revealed numerous
genes whose expression differs between IBC and non IBC tumors. Most of these genes linked to IBC are
regulated by NF-kB, and are associated with inflammation, proliferation, cell motility, invasion, and
angiogenesis [13, 14]. The genes regulated by these transcription factors have also been linked with
chemoresistance and radioresistance [15]; thus, agents that can block these pathways might inhibit growth of
IBC and sensitize the tumor to paclitaxel.
Although numerous inhibitors of NF-kB and STAT3 have been identified, none of them yet have been
approved for human use in part because of lack of safety. Our laboratory has identified a compound curcumin
(also called diferuloylmethane) from turmeric (Curcuma longa) which can inhibit both NF-kB [16] and STAT3
pathways [17]. In addition curcumin has been shown to downregulate HER2 [18] and EGFR signaling [19], also
linked to IBC. Curcumin has been found to upregulate p53 [20] but downregulate VEGF [21] and inhibit Rho-
mediated NF-kB signaling [22], also implicated in IBC. In addition, we have shown that curcumin can suppress
the proliferation of human breast cancer cells [23] and suppress the breast cancer metastasis to lung in
orthotopic mouse model [24]. Histone deacetylase enzyme, an enzyme linked to epigenetic changes and one
of the target for IBC [25], has also been shown to be inhibited by curcumin [26]. In addition, IL-6 that mediates
its effects through the activation of STAT3, is overexpressed in IBC cells [27] and curcumin is known to down-
regulate this pathway in most cancers [28]. All these reports indicate that curcumin may have potential in the
treatment of IBC.

Central hypothesis: Based on above background, we hypothesize that curcumin can inhibit the survival,
growth and metastasis of IBC; and further potentiate the effect of paclitaxel.

Specific aims:
Specific Aim 1. Determine whether curcumin can potentiates the apoptotic effects of paclitaxel against
IBC cells in culture.
 For this aim, we will first determine whether curcumin potentiates the effects of paclitaxel against IBC cells
by using a variety of assays.
 We will then examine the effect of curcumin on constitutive and paclitaxel-induced NF-B and STAT3
pathways in IBC cells.
 Finally, we will examine the effect of curcumin on various gene products linked to survival, proliferation,
invasion, and inflammation that are regulated by NF-B and STAT3 in IBC cells.

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Specific Aim 2. Determine whether curcumin potentiates the antitumor and antimetastatic effects of
paclitaxel in orthotopically implanted human IBC in nude mice.
 For this aim, we will first investigate the effect of curcumin alone and in combination with paclitaxel on the
growth of IBC tumors in mice.
 We will then examine the effect of curcumin on metastases of IBC to liver, lung, and spleen.
 Finally, we will examine the effect of curcumin on the overall survival of animals implanted with IBC tumors.
Specific Aim 3. Determine the mechanism by which curcumin alone and in combination with paclitaxel
exhibits antitumor effects against IBC tumors in mice.
 For this aim, we will first examine the effect of curcumin on proliferation (Ki67), angiogenesis (CD 31), and
inflammatory biomarkers (COX2, NF-B,
STAT3) in tumor tissues.
 Second, we will determine whether
curcumin affects the expression of gene
products involved in survival, proliferation,
angiogenesis, and invasion in tumor
tissues.
 Third, we will examine the bioavailability of
curcumin in serum and in tumor tissues.

Preliminary data: We examined the efficacy

of curcumin against IBC cell lines. As shown in
Fig. 1, curcumin inhibited the growth of SUM190, SUM149 and IBC3 in a dose dependent manner. Of note,
SUM190 and SUM149 were more sensitive to curcumin as compared to IBC3. Because curcumin inhibited the
growth of IBC cells, whether this polyphenol can inhibit the expression of proteins involved in tumorigenesis
was investigated. Curcumin inhibited
the expression of proteins involved in
survival (Mcl-1, XIAP, cIAP-1, cIAP-2,
cFLIP, Bcl-2, Bcl-xL, survivin),
proliferation (COX-2, c-myc), and
invasion (ICAM-1) in SUM149 cells
(Fig. 2). Because all these proteins are
regulated by NF-B, whether these
cells express constitutive NF-B was
investigated (Fig. 3, left). As expected,
SUM149 cells expressed constitutive
NF-B. Of note, non-IBC cell lines
exhibited little or complete absence of NF-B
(Fig. 3, left). Furthermore, curcumin suppressed
constitutive NF-B in these cells in a dose
dependent manner. These results suggest that
inhibition of NF-B in these cells might contribute
to the inhibition of IBC cell growth (Fig. 3, right).
We also investigated the possible mechanism
involved in the inhibitory effect of curcumin on
NF-B activation in IBC cells. Curcumin down-
regulated the expression of signaling molecules
involved in NF-B activation such as p-Akt, p-
IKK/ and p-IB (Fig. 4).

SIGNIFICANCE: These observations should show that curcumin potentiates the anti-tumor effects of paclitaxel
by inhibiting NF-B and STAT3 and their downstream targets, leading to the inhibition of proliferation,
angiogenesis and invasion. Because curcumin has been routinely consumed by Asian people for centuries
without any adverse effects, its safety in humans is well established. Moreover in countries such as India
where curcumin is consumed incidence of breast cancer is much lower than in the US (79/million vs

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660/million). Demonstrating that curcumin can potentiate the antitumor effects of paclitaxel against IBC would
have enormous implications for cancer patients because paclitaxel alone is ineffective and unsafe. Overall
these studies can form the basis for clinical trials with curcumin alone and in combination with
chemotherapeutic agents in IBC patients.

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